ABR-Affinity BioReagents's Frequently Asked Questions

This helpful FAQ will answer most of your technical questions regarding ABR-Affinity BioReagents products. If you cannot find an answer here, please use the technical questionnaires, or e-mail the technical support department directly. You will receive a reply from our technical support scientists within 24 hours. Check back here often as we are always adding to this FAQ.

ELISA Assays: Why am I obtaining high background in my ELISA procedures?

Immunohistochemistry: Why am I obtaining a weak, diffuse signal or no signal?

Immunohistochemistry: Why am I obtaining high background in my IHC procedures?

Immunoprecipitation: Why am I obtaining high background in my IP procedures?

Immunoprecipitation: Why is my target protein not being precipitated?

Storage: How should I store the antibody?

Western Blot: Why am I detecting non-specific bands on my Western blots?

Western Blot: Why am I not obtaining a signal on my Western blots?

Western Blot: Why am I obtaining high background staining on my Western blots?

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ELISA Assays: Why am I obtaining high background in my ELISA procedures?

1. Insufficient washing --Wash according to the protocol. Invert the plate on an absorbent tissue and tap if necessary to remove the liquid.
2. Incorrect incubation times --Follow set protocol.
3. Contamination --Use distilled/deionized water for diluting the stock reagents

Why am I experiencing weak signal or no color development?

1.  Improper storage.
2.  Reagents have expired.
3.  Wavelength-plate may have been read at the wrong wavelength.

Immunohistochemistry: Why am I obtaining a weak, diffuse signal or no signal?

1. The antibody being used may not be suitable for IHC procedures. Make certain that IHC is listed under Applications on our technical datasheet.
2. The fixation procedure (using formalin and paraformaldehyde fixatives) might be masking the epitope that the antibody recognizes. Try using antigen retrieval methods to unmask the epitope.
3. The primary and/or secondary antibody may be bad due to improper storage, improper dilution or excessive freezing and thawing. Be sure to run positive controls to ensure that the primary and/or secondary antibody is working properly.
4. Deparaffinization may be insufficient.  Deparafinize the sections longer and change out the xylene.
5. The protein is located in the nucleus and the antibody cannot penetrate the nucleus. Add a permeabilizing agent to the blocking buffer and antibody dilution buffer.
6. The PBS buffer may be contaminated with bacteria that damage the phosphate groups on the protein of interest.  Add 0.01% azide in the PBS antibody storage buffer or use fresh, sterile PBS.

Why am I obtaining high background in my IHC procedures?

1. The blocking serum used might be incorrect. Be sure to block according to the provided protocol and references.
2. Blocking might be insufficient. Increase the blocking incubation period to overnight at 4 C.
3. The primary antibody concentration being used is too high. Be sure to titrate the antibody to determine the optimal concentration.
4. The secondary antibody might be binding non-specifically. Make certain to run a secondary-only control.
5. Pre-absorb primary and secondary antibodies with 5% normal serum from the host species of the secondary antibody.
6. Endogenous peroxides may be active.  Add a quenching step to the protocol (hydrogen peroxide).

Immunoprecipitation: Why am I obtaining high background in my IP procedures?

1. The antibody concentration being used is too high. Be sure to titrate the antibody to determine the optimal concentration.
2. There is non-specific binding of other proteins to the affinity matrix being used. Be sure to preclear samples with affinity matrix before performing IP experiments.

Why is my target protein not being precipitated?

1. The antibody being used may not be suitable for immunoprecipitation procedures. Make certain that IP is listed under Applications on our technical datasheet.
2. The antibody being used may not be able to detect the species being tested. Make certain that the species being testing is listed under Applications on our technical datasheet.
3. Decrease your IP wash stringency. Sometimes stringent buffers (such as RIPA buffer) wash off the antigen or antibody. Try using PBS instead.
4. Make certain that the antibody being used is compatible with the affinity matrix being used (e.g. Protein A or Protein G bead type).

Storage: How should I store the antibody?

One should avoid repeated freeze/thaw cycles as antibodies can be adversely affected. It is usually preferable to store antibodies for a few days at 4°C rather than expose them to multiple freeze/thaw cycles.
DO NOT STORE ANTIBODIES IN FROST-FREE FREEZERS. These units subject the freezer compartment to regular freeze/thaw cycles to eliminate the frost on the sides of the freezer. At the same time, your antibodies are undergoing numerous freeze/thaw cycles.
Rather than repeatedly thawing and re-freezing the original unit, scientists should consider separating all or part of the unit into smaller aliquots. ABR-Affinity BioReagents recommends that you do not store antibodies in aliquots less than 20 ul, as this may cause the antibody to adsorb into the sides of the plastic vial. Instead, the researcher can perform an initial dilution (1:2 to 1:20) with a solution containing other proteins, such as BSA or non-fat dried milk. These additional proteins will act to stabilize the antibody in solution. Before diluting the antibody for storage the diluent should be sterile filtered to retard microbial growth.

Western Blot: Why am I detecting non-specific bands on my Western blots?

1. The concentration of primary antibody used might be excessive. Decrease the amount of primary antibody that is being used. Also, decrease the primary incubation period.
2. The secondary antibody might be binding additional proteins. Make certain to run a secondary-only control.
3. If available, use the blocking peptide to determine between specific and non-specific bands.
4. The protein may be forming multimers. Try boiling the samples for 10 minutes to disrupt the multimers.
5. The addition of a detergent (Tween-20) may help in reducing non-specific bands.
6. Cell lines that have been frequently passaged gradually accumulate differences in their protein expression profiles.  Go back to the original non-passaged cell line and run the current and original cell line samples in parallel.

Why am I not obtaining a signal on my Western blots?

1. The antigen may be deficient or may have been degraded. Be sure to load at least 25 µg of total cell lysate. Also, make certain that protease inhibitors are being used. Be sure to run positive controls to ensure that the transfer system is working.
2. The concentration of primary antibody may be insufficient. Use longer incubation times and higher concentrations of primary antibody. Be sure to run positive controls to ensure that the transfer system is working.
3. Do not over wash the membrane as excessive washing can wash away the primary antibody.
4. Do not re-use the primary antibody. It is possible that the concentration will decrease with each successive blot.
5. The primary antibody may be bad due to improper storage, improper dilution or excessive freezing and thawing. Be sure to run positive controls to ensure that the primary antibody is working properly.
6. The secondary antibody may be bad due to improper storage, improper dilution or excessive freezing and thawing. Be sure to run positive controls to ensure that the secondary antibody is working properly.
7. The antibody may not be able to detect the species being tested. Make certain that the species being testing is listed in the Species Reactivity on our technical datasheet.
8. Protein transfer may not have occurred. Be sure to use Ponceau S and Coomassie blue to make sure proteins were transferred from the gel.

Why am I obtaining high background staining on my Western blots?

1. The membrane may have been insufficiently blocked. Block membranes for longer periods or at higher temperatures. Also, increase the concentration of BSA or non-fat dry milk that is being used.
2. The concentration of primary antibody used might be excessive. Decrease the amount of primary antibody that is being used. Also, decrease the primary incubation period.
3. Washing of unbound antibodies may be insufficient. Increase wash periods and add 0.5% TWEEN 20 to wash solutions. Also, increase the concentration of salt.
4. When working with Phosphotyrosines, it is not recommended to use milk as a blocking reagent due to high concentrations of phosphoproteins.  BSA is a suitable alternative. 
5. PVDF membranes can cause background, although this type of membrane is recommended for high molecular weight proteins.