We'll sometimes provide an immunogen sequence, but it's not always publicly available.

Is the sequence information on datasheets?

For some antibodies, the immunogen sequence is widely known. In this case, you will see the immunogen information on the Overview tab of a product page, under "Immunogen" section. This section will also have the link to the UniProt database where the sequence of the target can be found.

What if the immunogen sequence is proprietary?

For other antibodies, the immunogen sequence won't be published, as the information can be commercially sensitive. We invest a lot of time and effort into selecting immunogen sequences to deliver the most effective antibodies. We sometimes decide to protect this information by giving this information on request to individual customers only.

If the sequence isn't available, but you need it for your experiments, please contact our Scientific Support team. For some externally sourced products, the immunogen sequence is proprietary information, and we are unable to release this information, even on request.

Can I get the epitope sequence?

Please note that the specific epitope will very rarely have been mapped for commercially available antibodies.

​The effect of incorrect storage conditions on a product depend on the product type, the temperature the product was stored at, and the duration of incorrect storage. Many products will still be viable if stored incorrectly for a short period of time. Unfortunately, we do not have exact guidelines for how long a product can be stored incorrectly.

We encourage you to run experiments on a small scale to see if the product works as expected, so you can decide whether to continue using it or discard it if it doesn't work.

A clone number is given to an antibody produced by a single clone of hybridoma cells.

Each clone number represents a specific cell line used to manufacture the antibody. Each cloned cell line receives a unique clone number.

The clone number differs from the lot number, which is related to the creation of the vial. There is little to no difference in the quality of the clones.

For monoclonal antibodies, the clone number can be found on the 'overview' tab of the product datasheet.

Are clone numbers different in the context of cell lines?

Clone also refers to an individual developed from a single somatic (non-germ) cell from a parent, representing a replica of that parent. A clone is a group of cells derived.

Answers to your frequently asked questions on PEG.

What is PEG?

PEG is the common abbreviation for polyethylene glycol – or, more properly, poly (ethylene glycol) – which refers to a chemical compound composed of repeating ethylene glycol units. PEG is an O-CH2-CH2 polymer, which is water-soluble, non-toxic, non-antigenic, and biocompatible. Purified PEG is most defined by their molecular weight (MW) ranges.

For example, PEG600 is PEG polymer with MW of 600.

PEG 10K or PEG 10,000 denotes a mixture of PEG molecules (about 195-265 PEG molecules) having an average MW of 10,000 g/mol.

PEG has several chemical properties that make it especially useful in various biological, chemical and pharmaceutical settings:

• Non-toxic and non-immunogenic – can be added to media and attached to surfaces and conjugated to molecules without interfering with cellular functions or target immunogenicities
• Hydrophilic (aqueous-soluble) – attachment to proteins and other biomolecules decreases aggregation and increases solubility
• Highly flexible – provides for surface treatment or bioconjugation without steric hindrance

Due to these unique properties, PEG is commonly used to PEGlyate various protein and drug molecules¹.

¹Veronese FM, et al. 2005. PEGylation, successful approach to drug delivery. Drug Discov Today. 10(21): 1451-8.

Why would I use an anti-PEG antibody?

An anti-PEG antibody can be used to monitor a drug’s pharmacokinetics, including distribution, metabolism, and excretio

Anti-PEG antibodies can also be used for the quality control of PEGylated proteins or molecules, such as nanoparticles. Our antibodies are suitable for use in ELISA, WB, and IHC.

What PEG products are available?

We offer various PEG antibodies as well as kits for the detection of PEG, including our high-affinity rabbit monoclonal PEG antibodies.

Anti-Polyethylene glycol antibody [PEG-B-47b] (Biotin) (ab53449)

• Recognises the PEG terminal methoxy group.
• ab51257 and ab53449 are the same clones (PEG-b-47). ab53449 is the biotinylated version of ab51257.
• ab53449 is purified before biotinylation. Protein A was used to purify this antibody.

PEG ELISA Kit (ab215546)

• Competitive quantitative ELISA kit.
• ab51257 is also used in the PEG ELISA kit (ab215546) as the capture antibody.

What are the smallest and largest PEG molecules your antibodies can detect?

Smallest detected - PEG 750
Largest detected - PEG 40K

Which PEG products do you recommend for detecting linear PEG and branched PEG?

ab51257 and ab53449 detect both linear and branched PEG based on our in-house ELISA results.

ab51257 is also used in the PEG ELISA kit (ab215546) as the capture antibody and therefore detects both linear and branched PEG.

Will your PEG antibodies work on my sample?

If there is a PEG molecule present, our anti-PEG antibodies will detect the PEGylated molecule. Our PEG products are independent of sample or species used.

What is the volume of PEG antibody per vial?

Our RabMAb PEG antibodies are provided as 100 ug.

You can calculate the volume using the following calculation:

(0.1mg) divided by the current concentration of antibody (mg/ml)= volume (ml)

How do I calculate PEG size using "N"?

The numbers that are often included in the names of PEGs indicate their average molecular weights, e.g. a PEG with n=9 would have an average molecular weight of approximately 400 daltons and would be labeled PEG 400.

Most PEGs include molecules with a distribution of molecular weights; i.e. they are polydisperse.

Therefore, N/Daltons is 9/400.  You can use this ratio to figure out either N or size in Dalton as long as you have either N or Dalton value.

What kind of buffer should be used to dilute my sample when using the PEG ELISA kit (ab215546)?

Use antigen/antibody diluent buffer in the kit. Any PBS-based buffer with BSA and/or Tween can be used.

What type of detection does the PEG ELISA kit (ab215546) use?

The PEG ELISA kit (ab215546) operates on the basis of competition between the enzyme (HRP) conjugated PEG and PEG labeled molecules for a limited number of binding sites on the surface of 96-wells coated with anti-PEG antibody.

The extent of color development resulted from the interaction between HRP and substrate TMB is inversely proportional to the amount of PEGylated molecules in the sample.

Is the PEG ELISA kit (ab215546) reactive with fluorocarbons containing PEG?

We have not tested fluorocarbons containing PEG so we do not know for certain. However, we tested H2N-PEG6000-OCH3 using the kit and it did not compete with HRP-PEG.

Is the PEG BSA reference sample provided in the PEG ELISA kit (ab215546) a single PEG conjugated to

The PEG BSA reference sample in this kit has about 5 moles of PEG5K conjugated to 1 mole of BSA.

To get the best results with our assay kits, we recommend reading the protocol book and the Precautions and Limitations, and Successfully Running Assays sections, before you start to prepare samples or reagents. For metabolite or enzyme activity assay kits, please also read the Sample Preparation and Experimental Data sections.

Is colorimetric or fluorometric detection more sensitive for microplate reader-based assays?

In general, fluorometric detection is about ten times more sensitive than colorimetric (spectrophotometric) detection. Check you have the right filters before buying a fluorometric kit.

Do you provide test samples?

Unfortunately, we do not offer test samples.

Assay kits are included in our Abcam trial program, for use in untested species or sample types without the financial risk.

At what temperature should I store the kit and what is the expiry date?

Storage temperatures are listed in the kit protocol book. If stored as recommended, assay kits can be stored for up to a year unless the protocol book says otherwise. Reconstituted components can typically only be stored for two to three months.

Can you disclose the composition of the buffers in your kits?

Unfortunately, we cannot disclose assay buffer constituents or concentrations as they are proprietary information. A SDS (Safety Data Sheet) on the components can be found on each product datasheet with information on any hazards.

Does Abcam sell kit components separately and can we use an alternate buffer for sample preparation?

In general, Abcam does not sell separate kit components, but some popular extra components are available. We highly recommend only using the buffers provided in the kit for the best results.

Do not mix components between different kits. If a lysis buffer is not provided, please follow the protocol recommended in our tissue and cell preparation guide.

What is your definition of a “test”?

Some kits are sold based on the number of “tests”. A "test" refers to a single assay well, and controls and standards necessary to perform the assay are also included in this number. The number of wells that contain sample, control or standard will vary depending on the kit.

In which species or samples will the kit work (cells, tissue, serum, blood, plasma, etc.)?

Our kits have been tested in human cell cultures/lysates (unless stated otherwise), and most kits have also been tested in mouse and rat samples. Read on to learn more.

Kits that detect generic metabolites (ammonia, carbohydrates, etc.) are expected to work in all species and their use is covered by the Abcam product promise. Due to the nature of the detection method of our enzymatic activity kits, we expect they will work with most mammalian species and with organisms such as bacteria, plants, drosophila or yeast; although this may require additional optimization. The sample types in which the kit has been tested are displayed on the product datasheet or protocol booklet.

If the sample you wish to analyze is not listed on the datasheet, please contact our  Scientific Support team. If you would like to try our kits in a non-tested species or sample, you can benefit from our Abcam trial program.

What are your recommendations for assaying multiple samples?

We recommend buying the same lot number when comparing or analyzing multiple samples. A new standard curve must always be prepared with each individual kit unit, however if ordering multiple kits from the same batch at the same time, we suggest comparing the standard curve of 2 kits, and if curves are identical, the same curve can then be used for other units of same batch.

Are kits ok to use when shipped over the weekend?

We recommend storing the kits at suggested temperature immediately after receiving. The shipping conditions over the weekend shouldn’t have any effect on quality.

How long do assays take?

The protocol times vary depending on the kit, analyte, and whether the samples require additional purification steps prior to measurement. If we are able to standardize the protocol time, this information will be included on the datasheet. However, be aware that this is only an indication, and you should plan your experiment carefully and after thoroughly reading the protocol.

Precautions and limitations of cellular and biochemical assays.

What safety precautions should I take when using or handling the kit?

All reagents should be handled with care and disposed of properly. Please review the Safety Datasheet (SDS) provided for information. Always observe good laboratory practice, including wearing gloves, a lab coat, and protective eyewear and not eating, drinking, or smoking in laboratory areas. All biological materials should be handled as if they are potentially hazardous and disposed of following established local safety procedures.

Can I use your products for diagnostic procedures?

No, our products are intended for research use only.

Are there any other materials required to use your kits which are not listed in the protocol?

Our kits require materials and tools commonly found in research laboratories, including MilliQ water or another type of double-distilled water (ddH2O), pipettes and pipette tips, including multi-channel pipettes, assorted glassware for the preparation of reagents and buffer solutions, and tubes for the preparation of reagents and buffer solutions.

Why do I have some leftover reagents after performing the assay?

Selected components in our kits are supplied with surplus amounts to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures.

Can I modify the protocol, or mix or substitute reagents or materials from other kit lots or vendors

The components of each kit have been carefully formulated and tested together on a lot-specific basis using the protocol provided. We understand that, occasionally, you might want to modify the protocol, extend the use of the kit by adding separate components from other kits, or use individual components in other assays. We cannot guarantee performance in these circumstances.

Including deproteinizing the sample, freezing and storing, and more.

Should I deproteinate my samples when using an enzymatic assay to measure a metabolite?

And which method should I use?

Detection of many metabolites such as NADH, NADPH or ATP can be hampered by the presence of interfering enzymes that would degrade the metabolite. By deproteinizing the sample, the interfering enzymes are removed and the stability of the metabolites improves.

For a quick and efficient deproteinization, we suggest using Deproteinizing Sample Preparation Kit – TCA (ab204708). This kit is based on using trichloroacetic acid (TCA) to precipitate proteins, followed by a neutralization step. This ensures precipitation of interfering proteins with minimal loss of metabolites.

Perchloric acid (PCA) is also commonly used for sample deproteinization; however, it is more dangerous and corrosive than TCA. For an alternative step-by-step protocol using PCA, follow our PCA-based Deproteinization Protocol.

Spin columns can also be used as a quick deproteinization step: metabolites are collected in the flow-through while enzymes/proteins are trapped in the filter. We recommend 10kD spin columns. It is possible to use smaller sized spin columns (such as 3kD) if the compound of interest has a small enough molecular weight to pass through the filter. Small-sized filters are hard to spin and dry, so these can result in a loss of metabolite. We recommend using 10kD spin columns only for liquid samples, as cell and tissue lysates will have debris that can block the filter.

Can I freeze and store my samples prior to analysis with enzyme activity and metabolite assays?

We recommend using fresh samples wherever possible for the best results, but we understand that this is not always possible due to experimental conditions. Read our suggestion:

Depending upon the analyte of interest we suggest deproteinization (typically required for metabolite assays), followed by snap freezing in liquid nitrogen, and storage at -80°C until use. Samples should undergo minimal freeze-thaw cycles (aliquot samples before freezing to avoid multiple freeze-thaws) and never be stored for long periods of time. This is extremely important for metabolites with short half-life or for proteolytic enzymes that can lose activity after storage.

How should tissues be prepared for analysis with enzyme activity and metabolite assays?

Tissue samples should be completely homogenized on ice in cold sample preparation buffer, preferably using a Dounce homogenizer. The amount of buffer necessary should be indicated on the protocol - read on to find out what to do if it's not.

If the amount of buffer isn't indicated, use between 4 - 6 times the tissue volume. Carefully transfer the homogenate to a tube (microcentrifuge, 15 mL or 50 mL tube depending on the amount of tissue) and centrifuge the sample at low speed in a cold centrifuge for 3 - 5 minutes. Transfer the supernatant to a new tube and discard the insoluble pellet. Read our tissue and cell lysate preparation guide for a general preparation procedure.

Failure to consistently and completely homogenize tissues can lead to erratic readings.
Some substances can interfere with assays and should be avoided in sample preparation. In general, substances such as EDTA (> 0.5mM), ascorbic acid (> 0.2%), sodium azide (> 0.2%), NP-40 and Tween-20 (>1%) interfere with many enzyme-based assays and should be avoided in sample preparation.

I don't have enough cells for your recommended starting amount. Can I scale down the buffers?

We've optimized each assay for the cell number stated in the protocol. You can try to scale down and decrease the amount of buffers used proportionally, but it may not fall within the range for the kit. This is why we encourage users to follow the sample recommendation in the protocol - if, for whatever reason, this isn't possible, we would recommend harvesting cells at different times and freezing them until there's enough sample to proceed with the protocol (this is not appropriate for assays to be run on live or fixed cells).

What is the exact volume of sample required for this assay?

We don't recommend a specific volume for the amount of any sample to be used since it's sample concentration and quality-based. We recommend running a pilot experiment with multiple dilutions to determine the optimal dilution that gives a reading within the linear range of the standard curve.

We recommend that for all assays, you consider the following to ensure that you get the best results:

How should I prepare reagents?

Our top tips:

• Avoid foaming or bubbles when mixing or reconstituting components.
• Avoid cross-contamination of samples or reagents by changing tips between sample, standard and reagent additions.
• Ensure plates are properly sealed or covered during incubation steps.
• Ensure all reagents and solutions are at the appropriate temperature for use before starting the assay (i.e., not taken straight from the ice bucket into the assay), and mix reagents gently before use.
• Don’t allow reagents to sit for extended periods before use, or reuse prepared reagents, always prepare fresh reaction mix before use.
• Make sure that all equipment is switched on and set at the appropriate temperature before starting the assay.

How do I perform the assay?

Our top tips:

• Ensure that you accurately follow the recommended incubation times and temperatures in the protocol.
• When preparing both reagents and samples and performing the assay, avoid pipetting small volumes (< 5 µL) and prepare a master mix where possible; this will reduce variability in results.
• Avoid air bubbles in wells, which can distort readings, by pipetting gently against the wall of the tubes.

How do I read the assay?

Our top tips:

• Carefully check that you are using the correct wavelength and filter settings for the assay.
• Ensure that you are using the right plate type:
  Colorimetric: clear plates
  Fluorometric: black wells/clear bottom plates
  Luminometric: white wells/clear bottom plates

How do I analyze assays?

Our top tips:

• Interfering substances in samples can distort results, read the protocol carefully to identify these and also deproteinize samples where recommended.
• For assays with a standard curve, with samples with analyte concentrations greater than the most concentrated standard, to obtain an accurate reading you should dilute the sample in the appropriate sample dilution buffer to bring it within the linear range of the standard curve.

Everything you need to know about processing your data.

For assays with a standard curve, how do I calculate my values?

You can find more details on how to calculate results back from the standard curve in the protocol booklet's Data Analysis section. Here's what to do if you need help:

If you need help generating a standard curve or using it to determine the concentration or value of your metabolite of interest in each sample, please check our helpful resource guide on calculating and evaluating ELISA data.

Why are my standard curve values lower than those shown on the datasheet?

Here are some factors that might influence the signals:

There are multiple factors that can influence the signals, like the incubation times, room temperature, handling, etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.

Why is sample normalization necessary?

When comparing the enzymatic activity of two or more samples or analyzing the effect of inhibitors or agonists on enzymatic activity between samples, it's always necessary to normalize the samples based on the number of cells or the protein concentration after cell lysis.

What are expected OD readings using sample X?

If we know of expected values using certain sample types, we'll add it to our datasheet.

What is the range of sensitivity of the assays?

Each assay has different sensitivities, depending on what it is measuring and the detection method used. We have added the information to the datasheet when available.

My microplate reader doesn't have the recommended filter. Can I still use the assay?

The filter recommended on the datasheet will detect the absorbance and/or fluorescence emission of the dyes and reagents at its maximum and optimal wavelength, which is very important if you have low amount of sample or weak enzyme activity. Filters that differ from the optimal wavelength +/-20 nm could still be used; however, the further they are from the peak the weaker the signal will be, which can lead to higher background and noise.

Find the answers to frequently asked questions about our ioSkeletal Myocytes.

What format will the cells be delivered to clients: frozen vials or pre-plated cells?

​Cells are provided as frozen vials, in either small (>2.5x10⁶ viable cells) or large (>5x10⁶ viable cells) sizes and shipped on dry ice. They should be immediately stored upon arrival in liquid nitrogen or ultra-low temperature freezers (-150^oC) until use.

Are ioSkeletal Myocytes fully differentiated?

​ioSkeletal Myocytes are not fully differentiated when the end-user receives them. ioSkeletal Myocytes are shipped as ‘primed’ skeletal myocytes that have been generated from human pluripotent stem cells using patented opti-ox cellular reprogramming technology. Cells are delivered in a cryopreserved format are programmed to rapidly mature upon revival in the recommended medium.

The protocol for cell generation is a three-phase process:

• Induction
• Stabilization for three days with doxycycline
• Maintenance during with the skeletal myocytes mature

Can you propagate ioSkeletal Myocytes once revived?

ioSkeletal Myocytes cannot be propagated or passaged further in culture because they have initiated reprogramming.

What seeding density do you recommend for the ioSkeletal Myocytes?

Skeletal myocyte cultures are obtained by plating ioSkeletal Myocytes at a minimum seeding density of 100,000 cells/cm². This may require optimization depending on the experiment and plate format. We do not advise seeding below 100,000 cells/cm².

How are cells cultivated?

Cells are cultivated in serum-free, chemically defined culture conditions, as a 2D monolayer on Geltrex coated TC dishes. They are cryopreserved in knockout serum replacement (CTS-grade) supplemented with 10% DMSO.

How soon after delivery can ioSkeletal Myocytes be used for experiments?

ioSkeletal Myocytes are delivered in a cryopreserved format and are programmed to rapidly mature upon revival in the recommended media. By day three post-revival, ioSkeletal Myocytes demonstrate classical myocyte morphology and express the myocyte genes DES, MYOG, and myosin heavy chain, as assessed by quantitative reverse transcription PCR (qRT-PCR). By day seven post-revival, skeletal myocytes demonstrate expression of GLUT4 in peri-nuclear regions and striations. Skeletal myocytes express the major proteins of myofilaments, including myosin heavy chain, desmin, dystrophin, and troponin. By day 10 post-revival, form-striated, multinucleated myocytes that contract in response to acetylcholine are present.

Why is opti-ox better than other methods of cellular reprogramming?

ioSkeletal Myocytes are derived from human-induced pluripotent stem cells (hiPSCs) using proprietary opti-ox technology (as described by Pawlowski et al in 2017), which relies on the precise genetic engineering of hiPSCs with the transcription factor(s) defining a specific cell identity. The opti-ox system enables unprecedented batch-to-batch reproducibility, homogeneity of differentiation, and scalability compared to classical approaches using non-targeted transgenesis (eg viral vectors). ioSkeletal Myocytes are easy to culture, and, within days of revival, convert into homogeneous and mature skeletal myocytes.

What were the cells of origin for ioSkeletal Myocytes?

ioSkeletal Myocytes are generated from hiPSCs.

The parental iPSC line has been derived from Caucasian white male dermal fibroblasts using the four retrovirally transduced Yamanaka factors:

Oct3/4
Sox2
Klf4
c-Myc

Do you use viral vectors to manufacture ioSkeletal Myocytes?

No, only recombinant DNA vectors are used to generate ioSkeletal Myocytes from the parental hiPSC line*.

*However, replication-deficient retroviral vectors (non-infectious) have been used for the reprogramming of the parental hiPSC line (characterized master line from dermal fibroblasts).

What is the host and transgene used to generate cells?

The host is human, and the transgene used to differentiate the hiPSCs towards ioSkeletal Myocytes is MYOD1.

The cells also express additional transgenes that are an integral part of the opti-ox system:

• PAC (puromycin resistance, prokaryote)
• NEO (neomycin resistance, prokaryote)
• rtTA (TetON system, prokaryote)

What quality control is performed on the ioSkeletal Myocytes?

ioSkeletal Myocytes production batches are tested for sterility, viability, and maturity acquisition over time by monitoring the expression of key genes by RT-qPCR. This includes monitoring for loss of pluripotency (OCT4 and NANOG), and acquisition of Myosin Heavy Chains (MYH2, MYH3, MYH8), Troponin (TNNT1), along with Desmin (DES), Dystrophin (DMD) and the skeletal myocyte transcription factor Myogenin (MYOG).

In addition, production batches are tested by immunocytochemistry for the expression of Myogenin, Desmin, Dystrophin and Myosin Heavy Chain. The genetic integrity of the parental hiPSC clone used for ioSkeletal Myocytes manufacturing is tested by G-banding karyotyping and array genome hybridisation (AGH). Absence of common human pathogens (Hepatitis B, Hepatitis C, HIV1, HIV2, HTLV 1, HTLV 2), mycoplasma, bacterial and fungal growth is confirmed by validated means.

How are the cell lines confirmed to be free from contamination?

We follow strict aseptic bio-banking procedures. Each manufactured cell lot is tested for sterility (microbial and fungal) and absence of mycoplasma infection (pan species) by industry-standard, validated means, post-thawing.

Are there any references for the ioSkeletal Myocytes?

Yes! Please see this publication describing the reprogramming of human iPSCs into skeletal myocytes by MYOD driven opti-ox cellular reprogramming:

Pawlowski, M., Ortmann, D., Bertero, A., Tavares, J.M., Pedersen, R.A., Vallier, L., and Kotter, M.R.N. Inducible and Deterministic Forward Programming of Human Pluripotent Stem Cells into Neurons, Skeletal Myocytes, and Oligodendrocytes. Stem Cell Reports  Vol. 8 803–812, (2017)

How can I contact you if I have a question?

If you have a question about our products or services, get in touch here.

Your biochemical questions answered. For anything not covered below, please contact scientific support.

What should biochemicals be used for, and who should use them?

All products in our biochemical range are potentially hazardous and are strictly for laboratory research and development use only:

They shouldn't be used for pharmaceutical, veterinary, household, agricultural, food, cosmetic, or any other human use and should only be used by scientifically qualified personnel, trained in laboratory procedures. Material Safety Datasheets are supplied with all of our products and contain advice regarding the safe handling of your product. However, due to the novel nature of our products, the potential hazards are not always known; it's your responsibility to ensure that all relevant safety precautions are taken at all times.

What dose of antagonist, agonist, or signaling tool should be used in vivo?

This is complex and depends on a number of factors.

This includes the method of administration (intravenous, intramuscular, or intraperitoneal), bioavailability, half-life, rates of hepatic and renal clearance, binding to proteins, drug interactions and tissue-specific distribution and accumulation. As we do not carry out any biological studies of our products in vivo, we therefore recommend that you review the published literature for dosage guidelines.

What dose of an antagonist, agonist, or signaling tool should be used in vitro?

The amount of product required depends on many factors:

This includes target accessibility, cell permeability, duration of incubation, and type of cells or assay used; it is best to survey the literature to determine the IC50, EC50, or Ki. For an inhibitor, if published Ki or, IC50 values are known, we recommend that you use five to 10 times higher than these values to maximally inhibit enzyme or receptor activity. If there are no published values, we recommend that you perform dose-response experiments (running appropriate controls) and use Michaelis-Menten kinetics to determine the Ki value.

How do I determine if a compound is cell-permeable?

There is no easy way of predicting if a product will be cell-permeable. Generally, charged molecules are not cell-permeable. However, modified phosphorylated compounds, such as mono- and dibutyryl cAMP, are cell-permeable. High molecular weight peptides are generally not cell-permeable under normal conditions.

What is Abcam's biochemical product purity and quality? How are they determined?

Our biochemicals are of very high purity, typically >98%. Chemical purity and quality are determined using a comprehensive range of techniques, including HPLC, chiral HPLC, NMR, microanalysis, optical rotation, TLC, and mass spectrometry. Details are provided on the Certificate of Analysis that accompanies each product.

What should I do if I can’t see any product in the vial?

Products sold in small quantities may not be readily visible, as they can coat the bottom or walls of the vial. When stabilizing your product, it's important that you ensure that the solvent comes into contact with all areas of the vial. Read on to learn more.

Freeze-dried products often cause concern as they are sometimes difficult to see with the naked eye. Typical freeze-dried products that you often expect to see are shown in the first example below. The vial on the left depicts a dyed example of a product and the vial on the right is an undyed product.

In this case, it is easy to see the product in the bottom of the container. However, freeze-dried products can also resemble a smear around the vial. The second example below shows several ways these products can be distributed around a vial. The left and middle vials are examples of a dyed product, which makes the process easier to visualize, and the vial on the right (an undyed product) demonstrates how, although the product is present, it may appear invisible.

Should different batches of a product look the same?

It is not unusual for different batches of the same product to vary slightly in appearance and color. However, this will not affect purity or quality as described on the batch-specific Certificate of Analysis and will not affect product performance.

What should I know about the molecular weights and molecular formulas?

The molecular weight and formula given on the product datasheet correspond to the chemical structure displayed there. However, molecular weights (and formulae) can vary slightly from batch to batch due to water composition or a change of salt. These changes will be indicated on the Certificate of Analysis that accompanies your product. The changes should not affect the biological activity of your product, but it is important that you take them into account when making solutions, so you should always use the batch molecular weight as stated on the Certificate of Analysis or the vial label.

What's the easiest way to identify a chemical?

To help you more easily identify a chemical, we provide CAS registry numbers for our products. CAS registry numbers are unique numerical identifiers for chemical substances. The Chemical Abstracts Service (CAS), a division of the American Chemical Society, assigns these identifiers to every chemical that has been described in the literature. Please note, that whilst these are given as accurately as possible on our website, they may not always reflect the level of hydration or salt of the product supplied.

How do I determine which product will work best?

To help you select your product, we provide a brief summary of the biological properties of our products, together with some suggested reading (references) that you may find useful. Please note, that whilst this information is given as accurately as possible on our website, it may not always reflect the latest findings and is not exhaustive. It is intended as a guide only - we recommend that you carry out your own search of the scientific literature for full details of the product's biological activity.

Can I cite Abcam Biochemicals in my publications?

Since we are often asked for examples of publications that cite Abcam biochemicals, we try to provide examples of these with our product data wherever possible. If you or your colleagues publish a paper that cites Abcam biochemicals as the source of one or more of your materials, please send us the details, and we'll send you a free gift!

How do I dissolve my product?

Stabilization instructions can be found on the Certificate of Analysis accompanying your product or on the website. Some products may be difficult to solubilize, and rapid stirring, warming in a water bath, or sonication of the solution may help. Solubility is temperature-dependent; cooling or freezing solutions may precipitate the product out of the solution. Therefore, it's important to ensure that your product is completely re-dissolved before use.

How do I make solutions using NaOH equivalents when dissolving amino acids?

Amino acids are often difficult to solubilize. A common technique is to use 1 molar equivalent (1eq) of sodium hydroxide (NaOH).

To dissolve compound in 1 equivalent of NaOH(aq.), the following formula can be used to calculate the volume of NaOH (aq) (VolNaOH) of concentration (ConcNaOH ) needed:

E.g for a product with a molecular weight of 197.13g, a mass of 5 mg, and using 100 mM NaOH:

How should I store my product?

We carry out regular stability tests of our products. However, due to the novel nature of many of our biochemicals, there is little published information on long-term product stability. You should always follow the recommendations stated on the Product Datasheet. However, we offer the following as a general guide:

Solid: Provided storage is as stated on the product vial and the vial is kept tightly sealed, the product can be stored for up to 6 months.
Solution: Wherever possible, you should prepare and use solutions on the same day. However, if you need to make up stock solutions in advance, we recommend that you store the solution as aliquots in tightly sealed vials at -20^°C. Generally, these will be usable for up to one month. Before use, and prior to opening the vial we recommend that you allow your product to equilibrate to room temperature for at least 1 hour.

What effects can storage have on product solubility?

Solubility is temperature-dependent. Cooling or freezing solutions may lead to precipitation of the product from the solution. It is therefore important to ensure that your product is completely re-dissolved before use.

Why are Abcam biochemicals sometimes shipped at room temperature?

Why are our biochemicals sometimes shipped at room temperature when the vial is labeled 'store at +4^°C or -20^°C'? Storage in the refrigerator or freezer is often recommended for long-term stability. If the product is shipped at ambient temperature, it is considered to be stable for the duration of shipping and normal handling. Upon receipt, you should then store it in the refrigerator or freezer (as indicated on the label). If you have any specific shipping requirements, please contact customer service.

Are variations in pack size or weight a cause for concern?

Unless explicitly stated on the Product Datasheet, the quantity of material supplied in our vials is not weighed to the accuracy required for direct solution preparation.

How much peptide is in my vial?

Abcam biochemical peptides are supplied either by net or gross weight, which will be indicated on the Certificate of Analysis accompanying the product.

While peptides for research use are typically > 95% pure by HPLC, the peptide content of the solid may typically range from 70 to 90% as peptides contain counter ions (e.g. acetate, trifluoroacetate) and residual moisture. The peptide content of each batch is shown on the Certificate of Analysis that accompanies each product.

Net peptide weight: For those peptides supplied by net peptide weight, these are weighed so that the amount of peptide received is exactly that stated on the vial. The actual amount of solid present in the vial will be dependent on the peptide content.
Gross weight: For those peptides supplied by gross weight, it is important that peptide content is taken into consideration. The peptide content is determined by amino acid analysis and is typically 70-90%. The net peptide weight is calculated by multiplying the peptide content by the gross weight. For example, a sample of peptide with gross weight of 5 mg and peptide content of 85% has a net peptide weight of 4.25 mg (5 mg x 0.85).

How should I handle lyophilized peptides?

Peptides should be stored at -20^°C for maximum stability and should not be stored in frost-free freezers, as variations in moisture and temperature may affect product stability. Peptides are often hygroscopic, which means they can absorb water. This may decrease stability and reduce the overall peptide content; to minimize this, allow peptide samples to equilibrate to room temperature in a desiccator for at least 1 hour prior to opening the vial and weighing the peptides. Weigh out the peptides quickly and reseal the bottle tightly.

How should I solubilize and store peptides?

Dissolve peptides in an appropriate buffer (acidic peptides in basic buffer and basic peptides in acidic buffer), and if necessary, sonicate briefly. Peptides containing Trp, Met, or Cys require special care to avoid oxidation, so oxygen-free water or reducing agents may be used. For storage, peptide solutions should be aliquoted and kept frozen at -20^°C. Most peptides containing Trp, Met, Cys, Asn, or Gln have a limited shelf life. Long-term storage is not recommended.

Here are answers to the most common questions about Alexa Fluor^® conjugated secondary antibodies, straight from our product and scientific support specialists.

​What temperature should I store the antibody at?

Storage at 4°C should not exceed one or two weeks. Upon arrival, briefly spin the antibody tube to pull down the solution and aliquot the secondary antibody solution. Aliquots should be stored at -20°C or -80°C.

Why should I aliquot the antibody?

Aliquoting minimizes the possibility of contamination and prevents the degradation of the antibody due to continuous freeze/thaw cycles. We recommend avoiding preparing aliquots smaller than 10 µl, as stock concentrations may be affected by the antibody's evaporation and adsorption onto the surface of the vial.

How should an aliquot be stored?

Aliquots should be stored at -20°C or -80°C in dark, low-protein-binding tubes. Prolonged exposure to light would compromise the antibody activity due to photo-bleaching of the fluorophores.

Can I dilute the antibody for storage?

Antibodies may be preferably diluted in the same buffer in which they are shipped (0.02% sodium azide, 1% BSA, 30% glycerol, PBS). Alternatively, you may dilute the antibody in PBS. However, we recommend storing antibodies in their concentrated form as extensive dilution may affect their stability.

What blocking solution should I use with the antibody?

The datasheet indicates the most appropriate blocking solution for your experiment. The blocking solution should be made fresh before each use. Common blocking agents for specific applications are listed below.

*Blocking agents should be fully dissolved before adding the antibody. Undissolved blocking agents may result in artifacts being generated during your experiments.

Which solution should I dilute the antibody in for sample incubation?

Diluting your antibody in blocking buffer for sample incubation may help minimize background. The sample may also be incubated with an antibody solution lacking a blocking agent such as TBST (Tris Buffered Saline with Tween^® 20). The results may vary, but if background is not an issue we recommend diluting the antibody in buffer with low concentrations of blocking agent.

How long should I incubate my sample for?

Incubation times will mostly depend on the expression of your protein of interest. Generally, WB and IF/ICC will only require a one-hour incubation for effective detection.

Will exposure to light during sample incubation affect my results?

Samples should be incubated in the dark when using Alexa Fluor^® conjugated secondary antibodies. Extensive exposure to light could result in photobleaching of the dye and thus affect secondary antibody conjugate performance.

What temperature should I incubate my sample at?

The temperature at which your sample should be incubated with the Alexa Fluor® conjugated secondary antibody will be usually dictated by the incubation time:

What is the optimal dilution for my antibody?

Dilution of the antibody for detection may vary depending on the application, the experimental conditions, and your specific target. A good starting point is to dilute the secondary antibody ten times more than your primary. However, a titration curve using serial dilutions of the secondary antibody should be made to determine the optimal dilution. Titration should mimic the conditions of the intended experiment.

How can I minimize background?

The intense fluorescence of Alexa Fluor^® conjugated secondary antibodies provides greater signal amplification than other similar dyes. Secondary antibodies are usually diluted in blocking buffer during sample incubation to minimize non-specific binding. Working at an optimal dilution of your secondary antibody will also help minimize background. Make sure you wash your sample extensively after incubation with the antibody. You should also be aware that long incubation times, such as overnight, may result in increased background.

Can I re-use the secondary antibody solution?

For optimal results, we do not recommend re-using Alexa Fluor^® conjugated secondary antibodies. If re-using the secondary antibody solution, fresh antibody should be added as the effective concentration is lowered after each use.

Are Alexa Fluor^® conjugated secondary antibodies applicable to multi-color imaging?

Our Alexa Fluor^® conjugated secondary antibodies are ideal for multiplexing. They have been extensively tested to ensure high specificity and sensitivity. Our broad range of Alexa Fluor^® conjugates target numerous species allowing for labeling of multiple antigens. See below a table representing our current Alexa Fluor® conjugated secondary antibodies and their characteristics.

^* Appearance of the solution in the vial.

^** Typical emission color seen through a conventional fluorescence microscope with appropriate filters.

^*** It is not possible to view the light emitted by near-IF fluorescent dyes since human vision is insensitive to light beyond approximately 650 nm.

Which Alexa Fluor^® conjugated secondary antibodies would you recommend for multi-color imaging?​

When planning a multiplex experiment, it is important to use Alexa Fluor^® conjugated secondary antibodies with distinct emission maximum (at least 40 nm difference). The graph below shows the emission spectra of each product we currently offer.

For optimal performance, try using Alexa Fluor^® conjugated secondary antibodies without spectral overlap. See below a table showing common combinations for double and triple immunostaining.

How can I minimize cross-reactivity in multiplex experiments?

Cross-reactivity is not expected when using primary antibodies raised in different species (eg mouse and rabbit) and secondary antibodies that specifically target each species. If using primary antibodies raised in the same species, they must be of different isotypes (eg IgG and IgM). Secondary antibodies that react with specific isotypes will prevent cross-reactivity. Our catalog includes a broad range of Alexa Fluor^® conjugated secondary antibodies targeting diverse species and isotypes.

What does pre-adsorbed mean?

​​Pre-adsorbed secondary antibodies are ideal for eliminating potential antibody species cross-reactivity. Pre-adsorption of the antibody should be performed using serum from the same species as your sample. Thus, species-adsorbed antibodies are less likely to interact with endogenous immunoglobulins, reducing significantly non-specific binding

We provide a wide range of pre-adsorbed Alexa Fluor® conjugated secondary antibodies.

We do not offer free samples for testing purposes. For this reason, we encourage you to learn as much about a product as you can before making a purchase. Our product overviews provide extensive information on our product ranges.

Under our Abcam Product Promise guarantee, if the product does not perform as described on our datasheet, contact our committed team who will work with you to find a suitable solution.

We have a range of initiatives to help you find the right research tools. These include:

Abcam trial program

If you're using a product in an untested species or application, you can let us know how it performed in your experiments and earn its full value back for a future purchase*.

Trial sized antibodies

We offer selected recombinant antibodies in smaller size vials for a reduced cost.

*Terms and conditions apply.

AM ester derivatives are cell-permeable, making them very useful in live cell studies. Modification of negative carboxlate by AM esters results in an uncharged, hydrophobic indicator or chelator that can permeate cell membranes.

Once inside the cell, intracellular esterases, found in almost all cell types, will hydrolyze the AM group. The resulting fluorescent indicator or chelator would then be contained inside the cell and accumulate.

What stock solutions should I make?

AM esters should be reconstituted using high-quality, anhydrous dimethylsulfoxide (DMSO). It is advisable to keep AM esters in as concentrated stock as possible (e.g. 1-10 mM) so that minimal amounts (ideally < 0.1%) of DMSO are present in the loading solution.

How do I store AM esters?

Wherever possible, you should prepare and use solutions on the same day. However, if you need to make up stock solutions in advance, we recommend that you store the solution as aliquots in tightly sealed vials at -20°C, protected from light and kept anhydrous.

Solvents like DMSO will readily take up moisture, leading to decomposition of the dye.

Can I use AM esters in vitro?

This is intended as an introduction only. Specific protocols for any particular dye and cell type should be obtained from the literature. Generally, AM esters are used at a final working concentration between 1-10 µM.

Incubation time typically ranges from 30 to 60 minutes. However incubation conditions can vary among cell types and among indicators and ideally should be optimized for each cell type.

It is best to use serum-free culture medium and incubate at room temperature or 37ºC (although loading is often better at room temperature).

Cells should be washed at least once with fresh serum-free culture medium to minimize extracellular background fluorescence. If serum-containing medium is used for loading, then the loading concentration of AM ester may need to be increased to compensate for binding of AM esters to serum proteins.

In some cases (for example where MW >1,000), the loaded cells may require a further incubation in medium without AM ester for 20-60 minutes to allow complete processing of the AM ester by intracellular esterases.

Which surfactants and buffers should I use?

A surfactant may be added to the loading medium to aid dispersal of the AM esters; it is typically used at a concentration of <0.1% (wt/vol). For loading, the required volumes of the DMSO stock solutions of the AM ester and of the surfactant should be premixed and then dispersed into aqueous medium for loading.

If the loading medium is buffered with bicarbonate, then loading should be done under a 5% CO2 atmosphere to prevent alkalinization of the medium through loss of CO2.