I had eluted some protein from plants from magnetic anti-Strep tag beads with 1.8ml of buffer with biotin (100 mM NaPhosphate, pH 8.0; 150 mM NaCl; 0.05% TX-100; 50 mM Biotin). When I added 100% TCA to a final TCA conc of 25% and kept it in the fridge for 30 mins and then -20 for another 30 mins, I found very quickly a large precipitate formed. It was very white and much more than I expected for the small amount of protein I was expecting. Is there anything obvious the precipitate could be, as it surely cannot be my protein of interest/interactors? I am new to protein work so I am just really confused and unsure what it could be (first time using TCA to precipitate protein).
its because TCA acts primarily through two mechanisms:
- Dehydration of the hydration shells around the protein: These are structured regions of water that form around hydrophobic patches on the surface of the folded protein. The waters in the shell are stolen into the hydration shell around the precipitant, effectively increasing the hydrophobic effect. This mechanism is in common with other precipitation agents, such as ammonium sulphate.
- In addition, the anionic TCA may trigger partial protein unfolding through disruption of the electrostatic interactions which determine the native tertiary structure of the protein. As a result, the usually well-hidden hydrophobic interior of the protein becomes exposed to the solvent.
I have done many TCA precipitations from eluted immunoprecipitates and the white pellet is always there; it has never been a problem. It would even be in my negative controls, yet the blots were always clean, so it must be something in the elution buffer that co-precipitates with the protein.
You can get a large ppt in case of crude proteins. How is the quality and quantity of the proteins on SDS-PAGE?
Raham Sher Khan So I was going to give the pellet to the MS service, based on protocol from collaborator. But now I am considering getting the large pellet into a solution and run it on a gel to see what it looks like and maybe send it for MS. But I am waiting on collaborators to approve of this action.
I am also planning on running a Western blot on the input and the elutions (pre-TCA), but I need to figure out how to run a Western first.
Sean Seltzer So I got my affinity purification protocol from a collabrator and when I showed them photos of my pellet they were surprised by how large it is. I do use a different type of bead to do the purification to them (I needed one for Strep and not FLAG). But other than that, I do not understand why I would get the precipritate when they do not (unless I had contamination from somewhere).
I was not going to run a gel and pass the pellet straight to go for MS, as my collabrators do, but I have suggested to them that I do run it on a gel to see what it looks like. But I am waiting on their approval right now.
James Peter Brook Lloyd You can always try adding the TCA to the biotin buffer alone and see if the buffer is causing the problem, if the pellets are as large as you describe; mine were about 5 uL in volume in a standard 1.5 mL tube. My protocol used FLAG beads and the elution was done with FLAG peptide in a similar buffer with NP40 instead of TX-100 and a protease inhibitor. I agree that running the IP on a gel would be the best thing. I think it will run well. As I also sent samples for MS, I would run a duplicate of the IP on a gel to confirm that it worked and was specific.
TCA is a relatively weak acid so it cannot hydrolyze the peptide bonds of proteins, but it does maintain an acidic pH in water. Addition of TCA to proteins in an aqueous solution disrupts the hydrogen-bonded water molecules (hydration sphere) surrounding a protein.
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