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Frequently asked questions and tips for success using our products, covering everything from conjugation kits to biochemicals, PEG, ioSkeletal Myocytes and more.
We'll sometimes provide an immunogen sequence, but it's not always publically available.
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.
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.
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.
A clone number is given to an antibody produced by a single clone of hybridoma cells.
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.
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.
An anti-PEG antibody can be used to monitor a drug’s pharmacokinetics, including distribution, metabolism, and excretio
We offer various PEG antibodies as well as kits for the detection of PEG, including our high-affinity rabbit monoclonal PEG antibodies.
Smallest detected - PEG 750
Largest detected - PEG 40K
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.
Our RabMAb PEG antibodies are provided as 100 ug.
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.
Use antigen/antibody diluent buffer in the kit. Any PBS-based buffer with BSA and/or Tween can be used.
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.
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.
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.
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.
Unfortunately, we do not offer test samples.
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.
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.
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.
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.
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.
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.
We recommend storing the kits at suggested temperature immediately after receiving. The shipping conditions over the weekend shouldn’t have any effect on quality.
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.
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.
No, our products are intended for research use only.
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.
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.
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.
And which method should I use?
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:
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.
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).
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:
Our top tips:
Our top tips:
Our top tips:
Our top tips:
Everything you need to know about processing your data.
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:
Here are some factors that might influence the signals:
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.
If we know of expected values using certain sample types, we'll add it to our datasheet.
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.
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.
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 (-150oC) until use.
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.
ioSkeletal Myocytes cannot be propagated or passaged further in culture because they have initiated reprogramming.
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.
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.
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.
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.
ioSkeletal Myocytes are generated from hiPSCs.
The host is human, and the transgene used to differentiate the hiPSCs towards ioSkeletal Myocytes is MYOD1.
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).
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.
Yes! Please see this publication describing the reprogramming of human iPSCs into skeletal myocytes by MYOD driven opti-ox cellular reprogramming:
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.
All products in our biochemical range are potentially hazardous and are strictly for laboratory research and development use only:
This is complex and depends on a number of factors.
The amount of product required depends on many factors:
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.
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.
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.
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.
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.
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.
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.
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!
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.
Amino acids are often difficult to solubilize. A common technique is to use 1 molar equivalent (1eq) of sodium hydroxide (NaOH).
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:
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 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.
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.
Abcam biochemical peptides are supplied either by net or gross weight, which will be indicated on the Certificate of Analysis accompanying the product.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The temperature at which your sample should be incubated with the Alexa Fluor® conjugated secondary antibody will be usually dictated by the incubation time:
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.
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.
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.
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.
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.
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.
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 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:
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.
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.
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.
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.
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.