FAQs about Catalog Antibodies
Is there any species preference for primary antibodies for western blot?
If an antibody works in immunolocalization, will it also work in a western blot and the other way around?
If an anti-peptide antibody detects a protein in a certain species, can I assume that it will work the same for all other species?
Is there any way to know if the commercial antibody I want to purchase will work on my species and in my application?
I bought an antibody and used it. I purchased it again and it does not work in the same way! How do you explain this?
I have purchased an antibody and get a lot of background bands in my western blots. Why?
I would like to re-use my primary antibody solution. Why is there no information about it on your product info sheets?
How long do antibodies last and how do I store them?
Q: Is there any species preference for primary antibodies for western blot?
A: 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?
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Q: If an antibody works in immunolocalization, will it also work in western blot and the other way around?
A: 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), and if such epitopes are not exposed following fixation, the antibody is not going to work.
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Q: If an anti-peptide antibody detects a protein in a certain species, can I assume that it will work the same for all other species?
A: 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 80% 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.
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Q: Is there any way to know if the commercial antibody I want to purchase will work on my species and in my application?
A: 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.
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Q: I bought an antibody and used it. I purchased it again and it does not work in the same way! How do you explain this?
A: There can be different reasons behind it:
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A: 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.
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Q: I have purchased an antibody and get a lot of background bands in my western blots. Why?
A: 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.
A: 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.
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Q: I would like to re-use my primary antibody solution. Why is there no information about it on your product info sheets?
A: 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.
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A: 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.
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Q: How long do antibodies last, and how do I store them?
A: 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.
More questions? Send us a message or check here:
Agrisera western blot protocol | Antibody storage | Video tutorial | Secondary antibodies | quantitative western blot-method| | quantitative western blot video tutorial
A: 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.
More questions? Send us a message or check here:
Agrisera western blot protocol | Antibody storage | Video tutorial | Secondary antibodies | quantitative western blot-method| | quantitative western blot video tutorial