Primary Antibodies: General FAQs

Frequently Asked Questions

  1. Is it okay to touch the gel (with gloves) when we are preparing the sandwich for transfer?
  2. I am interested in buying one of your antibodies. I am trying to place an order but your company is not on our system. Would you be able to tell me if you have any specific UK suppliers that you use?
  3. Does your company make custom antibodies?
  4. I have a question regarding the blocking buffer for western blotting. You said I shouldn't use milk as blocking buffer if I want to test the phosphoprotein, so what should I use as blocking buffer? Is it BSA alone with PBS TX?
  5. What is the purpose of using preservatives in antibodies? Does the preservative agent influence the antibody specification?
  6. Can primary antibodies be reused?
  7. What is the difference between cross adsorbed/normal/purified antibodies?
  8. I have a question regarding the blocking buffer for western blotting. You said I shouldn't use milk as blocking buffer if I want to test the phosphoprotein, so what should I use as blocking buffer? Is it BSA alone with PBS TX?
  9. Is your guarantee that you will refund the full price of the antibody if it doesn't work as specified still honored with this sample size?
  10. Does your company honor this guarantee if the antibody is purchased through a vendor?
  11. Can you please explain whether these two applications are the same: functional and blocking/neutralizing? If not, what's the difference?

Do you provide any epitope information for your antibodies?

It often will depend on the product how much epitope information the lab is willing to provide. In some cases we can provide the exact sequence and in others a range is usually available that the peptide falls within.

Do you have any recommendation for a blocking buffer for use of a primary antibody in Western Blot?

The blocking buffer that is often employed in our Western Blot QC validation is either 3% BSA, or 5% NFDM (Non-fat Dry Milk). In certain cases a mixture of both BSA and NFDM will be employed. Ensure that BSA is used as the blocking buffer for antibodies that target phosphorylated post translational modifications. This is to ensure that the antibody does not non-specifically bind the casein found in NFDM.

Can we use and IHC antibody for an ICC application?

On our datasheets if we state in the applications, Immunohistochemistry this means we have only validated the Antibody for use in this application. However, an antibody that has been validated to work in IHC is very likely to work in ICC. If you would like to test one of our antibodies that we have not yet tested for ICC you may like to participate in our Innovator’s Reward Program. Under the terms and conditions of this reward, by submitting a review on an untested application and/or species, regardless of positive or negative results, Novus will offer you a credit for the full price of the antibody. Additional details may be found here: (www.novusbio.com/support/innovators-reward.html).

Could you please provide information on how to find positive controls for your primary antibodies?

The images on our datasheets will provide detailed information on potential positive control cells and/or tissues, which have been previously tested by the lab for antibody validation. We also provide a related products tab at the top of the datasheet, which will contain useful control information where available. In addition to these tools, we recommend checking tools such as Uniprot or Protein Atlas to determine tissues and cell lines that highly express your protein of interest.

Is it okay to touch the gel (with gloves) when we are preparing the sandwich for transfer?

No, it is not recommended to touch gel or transfer membrane with bare hands and you should always handle it with the same the gloves on. Proteins or microorganisms present on hands may contaminate the gel or the membrane which results in a dirty blot or a blot with dark patches.

I am interested in buying one of your antibodies. I am trying to place an order but your company is not on our system. Would you be able to tell me if you have any specific UK suppliers that you use?

I am writing from our European office based in the Abingdon Science park. If you email info@bio-techne.com they will place your order.

Does your company make custom antibodies? 

Yes, we offer custom services through our parent company, Bio-Techne. Our expert scientists will work with you to deliver the antibody that meets all your required specifications. In tandem, an assigned project manager will ensure clear and constant communication throughout. We generate antibodies under controlled conditions with rigorous quality control testing to ensure high specificity and outstanding performance in the desired application(s). Please visit this webpage to learn more: www.rndsystems.com/rnd_page_objectname_custom_services.aspx

I have a question regarding the blocking buffer for western blotting. You said I shouldn't use milk as blocking buffer if I want to test the phosphoprotein, so what should I use as blocking buffer? Is it BSA alone with PBS TX?

As a general guideline in western blot to detect any phoshpho protein, it is usually recommended to use 5% w/v BSA (in TBST) simply because milk contains casein which is a phosphoprotein and as a result it can cause high background by detecting the casein present in milk.

What is the purpose of using preservatives in antibodies? Does the preservative agent influence the antibody specification?

The purpose of a preservative is to prolong the shelf-life of antibodies, so may be preferable for long-term storage. However, all our antibodies are guaranteed for 1 year from date of receipt, regardless of whether a preservative is present or not. Sodium azide specifically is used to inhibit bacterial growth. The preservative will not affect the specificity of the antibody (where it binds). Sometimes, it may be preferable to have an antibody without a preservative. For example, azide can interfere in antibody labeling reactions. It can also inhibit HRP (the enzyme label). Due to the toxicity of azide, its presence is not suitable for some applications, and furthermore high concentrations of azide are subject to regulations / health and safety warnings.

Can primary antibodies be reused?

We do not recommend that antibodies are reused.

What is the difference between cross adsorbed/normal/purified antibodies?

Pre-adsorption (also referred to as cross-adsorption) is an extra step to increase the specificity of an antibody. Antibodies (for example, antibodies recognizing rabbit IgG light-chains) are passed through a matrix containing immobilized serum proteins from potentially cross-reactive species (for example, antibodies that recognize sheep and bovine light-chains). Only antibodies specific to rabbit IgG-light chains will pass through the column. Antibodies cross-reacting with sheep or bovine IgG light-chains will bind and stay adsorbed to the matrix. The result of the procedure is a more specific antibody, which is especially useful in IHC when cross-reactivity is feared. Purified antibodies have been purified by binding to protein A or protein G or by binding to the immunogen used to generate the antibody.

I have a question regarding the blocking buffer for western blotting. You said I shouldn't use milk as blocking buffer if I want to test the phosphoprotein, so what should I use as blocking buffer? Is it BSA alone with PBS TX?

As a general guideline in western blot to detect any phoshpho protein, it is usually recommended to use 5% w/v BSA (in TBST) simply because milk contains casein which is a phosphoprotein and as a result it can cause high background by detecting the casein present in milk.

Is your guarantee that you will refund the full price of the antibody if it doesn't work as specified still honored with this sample size?

All of our products are backed by the 100% Novus Guarantee for the applications and species listed on the datasheet. Yes, this applies to sample sizes.

Does your company honor this guarantee if the antibody is purchased through a vendor?

All of our products are backed by the 100% Novus Guarantee for the applications and species listed on the datasheet. Yes, this guarnatee applies to products purchased through other vendors.

Can you please explain whether these two applications are the same: functional and blocking/neutralizing? If not, what's the difference?

The term "functional" is used to describe whether the protein has functional activity. This can be measured in a variety of ways, for example the ability of the protein to inhibit cell proliferation. If the protein has functional activity there is usually an ED50 value given on our product datasheets, so that our researchers have an idea of the concentration they should be using to see the desired effect. "Blocking" or "neutralizing" refers to the binding of an antibody to an antigen in order to prevent the antibody or antigen from exerting its effects. For example a peptide with blocking or neutralizing activity could be used to bind to an antibody thereby preventing it from binding to anything else. I just double checked this with my colleagues and to be on the safe side they said that the abbreviation depends on the product, and we would need to confirm for sure per product.