Q: Will this antibody cross-react?

<p>When selecting an antibody, it&#39;s important to ensure it is specific to the target and does not cross-react. Find out what you need to know to minimize cross-reactivity.</p>

Question 1

How do I determine protein concentration?

Accepted Answer

The quickest way to estimate the amount of protein in solution is to use UV-vis to measure absorbance directly, but this is generally not very accurate or sensitive. Highly accurate quantitation of most proteins can be achieved using either a Bradford or bicinchoninic acid (BCA) assay.

Question 2

How to use UV-vis to determine protein concentration?

Accepted Answer

Protein concentration can be estimated by measuring the UV absorbance at 280 nm; proteins show a strong peak here due to absorbance from Tryptophan and Tyrosine residues (commonly referred to as A280). This can readily be converted into the protein concentration using the Beer-Lambert law (see equation below). This method is used mostly for a very rough estimation of concentration, e.g. to check the success of purification.

A = ɛcl

A is the absorbance (e.g. A₂₈₀)
ɛ is the molar extinction coefficient, M^-1cm^-1 – this can be found from the literature
c is concentration in M
l is the path length in cm – the length of the cuvette / microplate

Question 3

How do I use the Bradford assay to determine protein concentration?

Accepted Answer

The Bradford assay is a colorimetric assay that relies on the binding of protein to Coomassie Brilliant Blue.

When the dye binds to protein in an acidic medium, a shift in absorption maximum occurs from 465 nm to 595 nm, with a change in color from brown to blue.
Unknown protein concentrations are estimated by reference to a standard curve generated from proteins of known concentration, typically bovine serum albumin (BSA)

Question 4

How do I use a BCA assay to determine protein concentration?

Accepted Answer

The BCA assay is a colorimetric assay that utilizes the reduction of Cu²⁺ ions by proteins, and subsequent binding of BCA.

The generated Cu+ ions form an intensely colored complex with the BCA reagent, that has a very strong absorbance at 562 nm
Unknown protein concentrations are estimated by reference to a standard curve generated from proteins of known concentration, typically bovine serum albumin (BSA)

Question 5

How should I measure samples from a microplate?

Accepted Answer

Many kits will detect samples using a standard microplate format. Your plate choice will depend upon the detection method. Please see below for a summary of the plate types we recommend for each type of reading:

Question 6

What are the benefits of top-reading vs. bottom reading?

Accepted Answer

Top-reading often provides a better signal-to-noise ratio. This reading can eliminate cross-talk between wells and reduce background fluorescence. Bottom reading, however, often provides better results when working with cell-based assays. This is because some assay components (e.g. oily buffers) can interfere with the excitation or emission of the fluorophore, making it impossible to read from the top of the plate.

Question 7

How do I convert rpm to g when using a centrifuge?

Accepted Answer

Revolutions per minute (rpm) and g-force (g) are both used to describe the force exerted by a centrifuge when you’re spinning a sample. In the scientific literature, these units are often used interchangeably. If you want to replicate somebody’s experiment, it’s useful to understand how to convert one to the other.

Question 8

How do I convert rpm to g?

Accepted Answer

If you know the g-force (g) and radius (r), calculate the rpm using the formula below:

Question 9

How do I convert g to rpm?

Accepted Answer

If you know the rpm and radius (r), calculate g using:
g = rpm2 x r x 1.118 x 10-5

Question 10

Will this antibody cross-react?

Accepted Answer

When selecting an antibody, it's important to ensure it is specific to the target and does not cross-react. Find out what you need to know to minimize cross-reactivity.

Question 11

What should I consider when selecting an antibody?

Accepted Answer

When selecting an antibody, consider the following:

When selecting an antibody:

Make sure it's validated to bind to the target. Check the 'overview' and 'target' sections of the datasheet.
If chosing a secondary antibody, make sure it's raised against the primary antibody in a different species, e.g. a mouse primary might require a donkey anti-mouse secondary.

Question 12

How do I avoid cross-reactivity with pre-absorption and fragments?

Accepted Answer

Find out how to avoid cross-reactivity in your experiment.

If a secondary antibody has been pre-adsorbed against potentially cross-reactive proteins, it will not react with those proteins. This increases specificity and minimizes cross-reactivity.
Using F(ab) and F(ab')₂ antibody fragments rather than full immunoglobulins can further minimize potential cross-reactivity between antibodies and cell-surface receptors.

Question 13

How can I check potential cross-reactivity for similar proteins?

Accepted Answer

If you have the immunogen sequence of your antibody, you can check the sequence alignment of the immunogen with other proteins the antibody could react with.

Several websites provide tools for calculating the percentage alignment, which are accessible from the EMBL-EBI website. When comparing the immunogen sequence and another protein sequence, an alignment score of 85 % or higher indicates the antibody may cross-react; the percentage alignment should ideally be much lower than this.

Question 14

How do I convert between different concentrations?

Accepted Answer

Converting between concentrations can be a headache. To make this easier, see the table below for some common units you might encounter when using one of our kits. You need to know the substance’s molecular weight (Mw) to convert between units based on mass and moles.

Question 15

What if concentration is presented per well rather than per volume?

Accepted Answer

Kit data is sometimes presented in molar units without a corresponding volume, e.g. nmol/well. To determine the concentration in this case, you need to divide by this value by the volume of solution in the well.

Check the kit’s protocol booklet for the volume to load into the well; this will be assay dependent.

Most microplate wells are loaded with a volume of around 50µL (which corresponds to 0.05 mL, or 5x10-5 dm3).

Question 16

How do I calculate the dynamic range of an ELISA?

Accepted Answer

The dynamic range of an ELISA is the range of antigen concentrations that can be measured accurately by the assay. Read on for more on how dynamic range is determined and how to interpret it for different sample types.

Question 17

How do I calculate dynamic range?

Accepted Answer

Dynamic range can be determined by plotting a standard curve of antigen concentration against the detection signal from the assay.

Measurements that are considered accurate, and therefore within the dynamic range, must show:

Low standard deviation between replicates of the standards
Strong correlation between the antigen concentration and the detection signal

Our ELISA Kit datasheets and protocols usually detail the dynamic ranges of experimental samples that have proven to be measured accurately by the assay. These dynamic ranges are generally presented in one of two ways: either as concentrations of the protein being detected or as dilutions (more common for samples like plasma or serum).

Question 18

How do I interpret dynamic range when presented as concentrations?

Accepted Answer

The protocol of Abcam’s Human Frataxin ELISA Kit (ab176112) includes a table, listing ranges of total protein concentrations of lysates of various cells lines that we expect to be within the dynamic range. Those concentrations were within the linear range of the standard curve when they were tested.

At the low end of the dynamic range, the ELISA could accurately detect a total frataxin concentration of 0.5 µg/mL
At the high end of the dynamic range, the ELISA could accurately detect a total frataxin concentration of 100 µg/mL
The dynamic range is therefore presented as 0.5 – 100 µg/mL

Question 19

How do I interpret dynamic range when presented as a range of dilutions or percentages?

Accepted Answer

The dynamic range of the concentrations other of sample types, such as serum and plasma are usually presented as dilutions, either as fractions or as percentages.

That is the case for Abcam’s TNF alpha ELISA Kit (ab208348). The protocol includes a table of ranges of plasma dilutions within the assay's dynamic range.

For plasma collected in heparin:

At the low end of the dynamic range, the ELISA could accurately detect the protein in dilutions equivalent to 1/16 (6.25/100, or 6.25 %).

At the high end of the dynamic range, the ELISA could accurately detect the protein in undiluted plasma (100/100 =1, or 100%)

The dynamic range is therefore presented as 6.25 – 100%

Question 20

How do I calculate values from a standard curve?

Accepted Answer

A standard curve is used to accurately determine the concentration of your sample from the signal generated by an assay. The signal is never perfectly proportional to the sample concentration. A standard curve is designed to correct for these effects, so you know which concentration a given signal value corresponds to.

Question 21

How do I cite Abcam products in my published research?

Accepted Answer

Information on how to cite our products and leave reviews.

Question 22

What benefits are there to citing Abcam products in my published research?

Accepted Answer

Properly cited published research empowers other scientists.

Research and discovery progresses at an incredible speed when others can reproduce experiments from publications. A study that analyzed journal articles found that 54% of resources are not uniquely identifiable in publications, regardless of domain, journal impact factor, or reporting requirements (1). Because of this, it's often difficult for scientists to repeat successful protocols and expand upon published research.

Our References team searches through past and recent publications to provide the most accurate and current data on each datasheet. References can be found on our datasheets under the references tab.

Question 23

How do I cite Abcam in my paper?

Accepted Answer

To ensure the product can easily be identified and the results can be reproduced, make sure to:

State Abcam as the source of the antibody
Include the catalog abID
Mention the tested applications and dilution range
Mention the species samples experimented on

We ask that if you are publishing with an Abcam product you please email technical@abcam.com with a copy of your paper and the product used.

Question 24

Where do the star ratings on Abcam's products come from?

Accepted Answer

Our Abcam product reviews initiative ensures that when scientists review our products, we publicly display all feedback, positive and negative, on our datasheets in order to ensure the most honest data is available to the research community.

Question 25

Why should I leave an Abcam prodcut review?

Accepted Answer

Not only will you be helping the wider research community to find the best quality products, but you'll also be earning points. Your Abcam points will be added to your account with each public review you leave and can be redeemed on future orders with us.

Question 26

How do I submit a product review?

Accepted Answer

To find out how to leave a review and earn points, head to our product reviews page.

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