Catalog Antibodies FAQ
It does not matter from which species the primary antibody comes from for a western blot application. There are many good secondary antibodies available for most species. What is important is which part of a protein an antibody is made to. Is it going to recognize epitopes (stretches of 5-6 amino acids), which are exposed on a given protein after transfer to a membrane? Depending on the applied condition, some proteins can refold following the transfer, which can make a given epitope inaccessible. It also depends on if the western blot is performed in native or denatured conditions. The addition of 6-8 M urea to your loading buffer might help to keep your protein unfolded and accessible for an antibody to bind. Before you use an antibody in western blot, please check to which part of the protein it was made. Are there any references confirming the use of this antibody for this specific technique?
It depends on the antibody binding site on the protein. Is this part exposed after fixation or following the western blot? If a polyclonal antibody is made to a synthetic peptide, it recognizes a pool of epitopes (linear epitopes are streches of 5-6 amino acids). If such epitopes are not exposed following fixation, the antibody is not going to work.
Not really, it all depends on the conservation level of the specific peptide used to elicit this antibody. It can be checked by comparing the peptide used to elicit the antibody in question, to the sequence of your protein. The conservation level between the peptide used to make an antibody, and the peptide found in your protein seqence, needs to be around 70% to allow an anti-peptide antibody to work, but may be lower for antibodies made to larger parts of a protein. In some cases, 6-8 M urea needs to be included to fully unfold the protein. The electrophoretic mobility of a protein can also vary between different species.
Yes, to a certain extent it is possible to predict the outcome before making actual experiments. What is worth checking is the conservation level of the peptide or protein sequence used to elicit a given antibody, to the protein you are planning to detect. If a sequence is not provided on our website, please send us an inquiry about this, together with the sequence of the protein you are aiming to detect.
There can be different reasons behind it:
- You obtained another batch in the second purchase. Please, check it on the tube which contains a specific lot number. Each antibody batch is unique and can vary in its binding properties.
- Another secondary antibodies was used.
- Another reagent was used.
- Another set of samples were used.
Have you checked the recommended conditions to use for this specific antibody, included on the product info sheet? Some antibodies will work reasonably well and give good results regardless of the applied conditions, like membrane type, load per well and developing reagent. Some antibodies require a bit more consideration. Less background can be obtained for some antibodies when using a PVDF instead of nitrocellulose membrane for transfer, or by applying the recommended blocking. There are also variations between secondary antibodies. However, the most important factor to consider when evaluating the western blot results, is often the type of sample being analyzed. For more information, you can either contact us, or check out our Western blot troubleshooting guide, found here.
When attempting to do quantification, it is generally not recommended to re-use an antibody solution, as this may give non-consistent results. Therefore, such information is not included in our product info sheets. Every antibody is different, and the re-use approach might work for one antibody, but give weaker and weaker results for another. This is due to a certain amount of antibody being depleted in every performed incubation. You need to try this approach in your particular set-up to know if it is viable. But please keep in mind that the results might not be consistent. If you would like to save your primary antibody, you could consider using a more sensisitive detection reagent instead.
Each antibody is different. Some might last for 20-40 years, while others may only last a year. Storage conditions are of importance to extend the antibody lifespan, but the stability of each specific antibody might be different. Therefore, please follow the information provided on the specific product information sheet for each antibody. What works for one antibody, might not necessarily be applicable for another. This is very important to keep in mind. If iyou have further questions, please contact us. General recommendations for antibody storage can be found here.
One big difference between antibodies offered in the formats serum, total immunoglobulin fraction and antigen-purified, is the amount of specific antibodies per µl or µg.
When calculating how many experiments an antibody can be used for, one should always check the suggested antibody dilution in the context of amount of protein/well in Western blot, not how many µl or µg of antibody is offered in one tube.
Below is a table showing the amount of specific antibodies in different formats.
When calculating how many experiments an antibody can be used for, one should always check the suggested antibody dilution in the context of amount of protein/well in Western blot, not how many µl or µg of antibody is offered in one tube.
Below is a table showing the amount of specific antibodies in different formats.
| Antibody format | Specific antibody amount |
| Serum | Unknown |
| Total Immunoglobulin fraction | 0.5-5% |
| Antigen-purified antibody | >95% |
