Hello!
I have produced monoclonal antibodies by transfecting HEK293 cells and purifying then using a protein A column.
To check for purity I ran several SDS-PAGEs and saw faint bands at ca 70 and 100 kDa in addition to the expected bands (see the example attached).
I suspected chaperones, and after mass spec gave me hits on HSP70 and HSP90 I am quite sure that I am mainly dealing with HSP70 (and some HSP90) being stuck to my antibodies somehow.
How can I get rid of them, assuming that they are bound to the antibodies?
Does this mean that the antibodies are misfolded?
Have you tried a size-exclusion chromatography technic? I suggest you try that to ensure the unwanted proteins are not bound to the antibodies.
Hello Julien Dembélé!
Thank you for your interest.
I have run SEC, in addition to running an ELISA with an HSP70-binding protein and a secondary antibody against human IgG.
The SEC shows one single peak, and the ELISA was positive.
I am in other words quite sure that the unwanted proteins indeed are bound to my antibody, and that the protein is HSP70.
My question is: is there any established technique to remove HSP70 when it is stuck to purified protein?
Hi Johanna,
A former colleague of mine added 5 mM of ATP to his washing buffer during affinity purification to remove HSPs from his protein. Binding of ATP supposedly switches HSP70 to a low-affinity state, thus removing it from your protein of interest.
See this article for more information: Article Mayer M, Bukau BHsp70 chaperones: Cellular functions and mol...
While I can't confirm myself that this step is working, it might be helpful information for you.
Cheers,
Jan
Hello Jan Philipp Dobert!
Thank you for the input. I have read about the same procedure (often in combination with Mg), but these articles were always dealing with the bacterial homolog DnaK from E. coli (this one for example: Article Removal of DnaK contamination during fusion protein purifications
)The above-mentioned article also sees improvements when you add denatured proteins, which can act as high-affinity substrates for the HSP. Maybe that's the way to go..
Johanna
1. Improve cell culture and expression conditions to reduce the amount of heat shock proteins produced. 2. Use affinity chromatography to purify the monoclonal antibody, as heat shock proteins have a high affinity for certain resins which can be used to separate them from the desired product. 3. Utilize size exclusion chromatography to remove low molecular weight heat shock proteins that can't be removed by affinity chromatography. 4. Utilize ion exchange chromatography, as this technique can be used to separate heat shock proteins based on their isoelectric point. 5. Utilize hydrophobic interaction chromatography, as this technique can be used to separate heat shock proteins based on their hydrophobicity. 6. Use a combination of various chromatographic techniques to maximize the removal of heat shock proteins from the desired product.
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