I am doing a project with biotinylated proteins. I am trying to purify only the biotinylated proteins from a conditioned cell culture media. I use Q-sepharose as my first step, followed by PD-10 to get my proteins into PBS. Then I concentrate it, then run it through the Streptavidin agarose (SA). I elute by boiling in SDS-PAGE buffer for ten minutes. Western blots with an anti-biotin antibody show good bands from the pre-SA and flow through of SA, but none from the "purified" sample. Can anyone tell me why my proteins are not binding?
Hi there,
Do preSA, FT and purified samples go exactly through the same treatment before loading on gel?
If the protein is in the FT it means it doesn't interact with beads. The obvious reason would be because of structural hindrance (the biotin being for whatever reason out of reach of streptavidin).
There are several reasons why your biotinylated proteins may not be binding to the streptavidin agarose:
Incorrect biotinylation: It is possible that the biotinylation reaction did not go to completion, or that some of the biotinylated proteins were cleaved or denatured during the purification process, which would affect the ability of the biotinylated moiety to bind to streptavidin.
Insufficient protein concentration: The concentration of biotinylated proteins in your sample may be too low for efficient binding to the streptavidin agarose, resulting in reduced binding and recovery of the biotinylated proteins.
pH issues: The pH of your buffer may be affecting the binding affinity of the biotin-streptavidin interaction. A pH between 7.2 and 8.0 is optimal for the biotin-streptavidin interaction, and a pH outside of this range can reduce binding efficiency.
Incomplete washing: The washing steps between the various resins are critical to removing unbound proteins and reducing background binding. If your washing steps are not thorough enough, unbound proteins can carry over and interfere with the binding of biotinylated proteins to the streptavidin agarose.
Contaminants: Presence of contaminants such as detergents, salts, or other proteins can also interfere with the binding of biotinylated proteins to streptavidin agarose.
To resolve these issues, I would suggest optimizing the conditions of your biotinylation reaction, increasing the protein concentration of your sample, adjusting the pH of your buffer, thoroughly washing the beads between steps, and minimizing the presence of contaminants. Additionally, you may consider using alternative purification methods, such as magnetic beads or column chromatography, to help improve the purity and yield of your biotinylated proteins
Streptavidin-biotin binding interaction is the strongest non-covalent interaction between two biological molecules. It is very difficult if not impossible to disrupt the bond between SA and biotinylated peptide, even after boiling with SDS. I would suggest using your antibiotin Ab to purify your biotinylated protein
Mary Cristine Charlesworth Can you point me in the direction of a protocol for purifying protein using the antibody?
I have assumed that your antibody is monoclonal or affinity-purified polyclonal.
You can ask the company that you buy the Ab from if they will covalently conjugate to beads (best way), or you can bind the Ab to Protein G/A magnetic beads, then incubate with your protein. You can use a mild acid like 5% acetic to release the protein from the bead, but you will also have Ab in your final sample. The advantage to covalently linked Ab is no Ab in the final sample.
There are a lot of protocols out there for IP of proteins using Protein A/G magnetic beads. The beads come with a protocol which would be a good starting point.
most probably your streptavidin agarose is dead... or you have free biotin in the media that saturate the streptavidin agarose and impairs protein binding...
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