TCA ppt of dansylated proteins
TCA ppt of dansylated proteins

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

Sample has about 1mg/ml of each of 3 dansylated proteins (Bovine serum albumin, ovalbumin, ribonuclease). Basic protocol is to dilute these in DW, add a carrier (sarkosyl, 0.05% final), ppt with TCA (10% final, wash with THF (tetrahydrofuran), dissolve in 0.1% SDS (0.1ml) with Tris base to neutralize TCA and observe by UV transillumination.

1 - 4 samples with 1,2,4, 10 microliters of protein add to 1ml DW, mix

2 - Add 10 microliters 2% sarkosyl (carrier), 5 microliters 0.1% Sudan Black (in acetone), mix
Sarkosyl solution should be < 2 wks old. Weigh 20mg aliquots into vials. Dissolve as needed
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 30 sec at 5K, decant supernatant, allow ppt to dry

7 - Dissolve in 100 microliters 0.1% SDS with 1 microliter of 1M Tris, observe by UV transillumination

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