Larger scale TCA ppt of proteins
Larger scale TCA ppt of proteins
Method is basically that of Chevallet, et al Proteomics 7, 1757-70 (2007)
1 - To 5ml of dilute protein (screw cap conical tube) add:
2 - Add 40 microliters 2% sarkosyl (carrier), 9 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 30 microliters 1mg/ml protamine sulfate (basic protein, carrier), 5 microliters 0.1% Ponceau S (stains basic proteins), mix.
3 - Add 450 microliters 100% TCA to each, mix, store 10 min in ice bath
4 - Centrifuge 3 min at 4.5K in S4180 rotor (Allegra centarifuge)
5 - Decant supernatant carefully, tap out excess solvent
6 - Wash with 150 microliters THF (tetrahydrofuran - in hood), centrifuge 3 min at 4.5K, decant supernatant, allow ppt to dry
7 - Dissolve pellet in (30 microliters) borate cracking buffer/w 1mM iodoacetamide
8 - Transfer to microfuge tube and heat 4 min @ 100C.
9 - Run 4, 12 microliter aliquots
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.