TCA ppt of proteins
TCA ppt of proteins

Method is basically that of Chevallet, et al Proteomics 7, 1757-70 (2007)

1 - To 1ml of dilute protein add:

2 - Add 10 microliters 2% sarkosyl (carrier), 3 microliters 0.1% Sudan Black (in acetone), mix
Sarkosyl solution should be < 2 wks old. Weigh 20mg aliquots into vials. Dissolve as needed
(2a) - Add 5 microliters 1mg/ml protamine sulfate (basic protein, carrier), 5 microliters 0.1% Ponceau S (stains basic proteins), mix.

3 - Add 90 microliters 100% TCA to each, mix, store 10 min in ice bath

4 - Centrifuge 30 sec at 13K in microfuge (in fridge)

5 - Decant supernatant carefully, tap out excess solvent

6 - Wash with 150 microliters THF (tetrahydrofuran - in hood), centrifuge 3 min at 13K, decant supernatant, allow ppt to dry

7 - Dissolve pellet in (30 microliters) borate cracking buffer/w 1mM iodoacetamide

Note: THF is miscible with water (compare THF properties to those of ether)

Note: Detergents, THF, or both should overcome effects of slip agents such as erucamide which commonly contaminate plastic centrifuge tubes.