I ordered a custom biotin-tagged polypeptide with 21 amino acids from the company. HPLC and MS show that the biotin tag is linked to the peptide. But I cannot detect biotin signals using western. I use another peptide from my colleague as positive control, and it works fine. Now I am thinking about switching to another company. But I am afraid of the some problem. Does anyone have some experience in this subject, what is wrong with the peptide, can anyone recommand a company with good custome peptide?
Thanks in advance!
Hi Zhiming: Assuming that your biotinylated peptide dissolved in water, and used the similar molar concentration as the positive peptide. Then, what's the probing antibody (avidin-HRP or streptavidin-HRP) you used to detect your biotin-peptide? I used the later (Pierce #21126) and found it very sensitive. Also, have you tried to load your peptide in the loading buffer containing only SDS and no heating? That may improve your chance of success. Good luck.
There may be two problems one pH and another detection probe or an antibody. Lower down the pH and use avidin-HRP in higher concenration. Keep the contents at 10-15 oC for 20 minutes.
This is because of high sensitivity of peptide, you just reduce analytical concentration and give a try, or otherwise the company will responsible for this, they may given a non-biotin peptide. company will responsible ask analytical report of your product.
Contact us, we will provide all custom peptides with full technical and research support with acceptable prize,
Grey Matter Research Foundation
www.gmrfoundation.com
Hi Zhiming:
Hopefully, biotin moiety is attached to your peptide via lysine side chain, or via 6-aminohexanoic acid residue, which guarantees its accessibility for avidin. Otherwise, steric restriction imposed by nearest residues may be an issue.
Best regards
Igor's point is well taken. All other suggestions are based on the assumption that the biotin moiety is freely accessible.
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