Hi! What could be the reason why, after I add 1 volume of Trichloroacetic acid (TCA) to 4 volumes of my sample (an elution fraction from size exclusion chrom), I incubate 10 mins at 4°C and I centrifuge 14K rpm 5 mins, I don´t see any pellet to proceed with this protocol I found online: TCA_ppt_protocol.pdf (caltech.edu) ? Too low protein concentration ?
I need to precipitate my protein for MS analysis.
Thank you!
Lavinia
TCA precipitation is less efficient at low protein concentrations. Also, very small protein pellets may be difficult to see. Including sodium deoxycholate with the TCA can improve the yield (1/10 volume of 0.15% sodium deoxycholate and 1/10 volume of 72% TCA), but you may prefer to avoid adding detergent if the sample is going to go for mass spec.
Add 100% (w/v) TCA (trichloroacetic acid, to the sample to bring the TCA concentration to 20%. Incubate on ice for at least 1 hr. Diluted samples may be left overnight. Spin at maximum speed in a microcentrifuge for 10 min. Wash the pellet 3X with ice-cold 0.01 M HCl / 90% acetone solution. Allow the pellet to air dry. The pellet can then be resuspended directly in 100 mM Tris, pH 8.5, 8 M urea, or 6 M Guanidine- HCl in 25 mM ammonium bicarbonate buffer for enzymatic digestion for mass spectrometry.
You may use Sodium DOC for solubilization, it is a mass spec compatible detergent if you can acidify the sample. At low pH, DOC will degrade, and the resulting degradation products can be removed by consecutive centrifugation. Thus, superantant is injectable. You can also get rid of excess DOC by performing an ethyl acetate LLE approach.
Hi Lavinia,
1. You can use 90% ammonium sulfate fractionation at 4°C. Most proteins are fractionated at 90% ammonium sulfate saturation at a particular temperature. However, desalination is an additional step in this process to obtain desalinated proteins. The ammonium sulfate concentration may be set by following this calculator
https://www.encorbio.com/protocols/AM-SO4.htm
2. You can use 2-3 volumes of chilled ethyl alcohol to protein sample at 4°C for precipitation of total protein. slowly add ethyl alcohol with gentle stirring. after the addition of ethyl alcohol (2-3 volume from the sample), the sample should be transferred to settle precipitate for a few h or overnight then centrifuged. Collect the pellet and resuspended it in the desired solvent.
I hope both techniques shall help you very well
İsmail Emir Akyildiz how do you add 100% TCA, if TCA is solid?
I wouldn't recommend ammonium sulphate precipitation. Reaching even 80% is pretty difficult and it works best in larger volume, which I imagine Lavinia Prosseda doesn't have, if she works with elution fractions from chromatography. Moreover, it also doesn't work that great at low protein concentrations, so that wouldn't be much better.
Lavinia, the problem may be either that the precipitation doesn't work because of low concentration or that your pellet is too small to be visible (or most likely combination of both). You can concentrate your sample using one of many centrifugal filters, if you don't mind that you will lose LMW peptides (how much depends on the cut-off). Alternatively, you may try "purification" and concentration on C18 columns, but I think large proteins bind too strongly and are impossible to elute.
What is your MS analysis protocol? Do you do digest or analysis of full-length proteins?
Tomáš Hluska,
Prepare 800 mL of distilled water in a suitable container. Add 1101.32 g of Trichloroacetic acid (TCA) to the solution. Add distilled water until the volume is 1 L. The resulting solution will contain 100% (w/v) TCA. TCA is highly hygroscopic...
bring the TCA concentration to 20% by mixing with the sample by applying a 1(TCA)/4 (sample) ratio dilution.
Lavinia Prosseda,
You may also try the Bligh&dyer approach to obtain protein pellet. Methanol-chloroform partition will result in protein floc which is a very efficient way to isolate proteome but also serves as an intermediate clean-up. On the other hand, a non-destructive/non-denaturating way of proteome isolation is the ultrafiltration physical method.
I left my samples at -4°C with TCA overnight and I could see a very faint pellet, like if the eppendorf was dirty. I added some loading buffer, ran a SDS-PAGE and I could see proteins by Comassie staining. So I guess the protocol works but my protein is very low concentrated, which doesn´t seem to matter to the MS facility.
Thank you all for your suggestions!
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