I´m a student from germany!
I´m trying to precipitate whole cell protein to get a higher concentration of my proteins for WB.
I use RIPA-buffer for cell lysis:
150 mM NaCl
50 mM Tris (pH 7,5)
0,5% sodium deoxycholate
Protease inhibitor cocktail tablet
2 mM EDTA
1 µg/ml Pepstatin
50 mM sodium fluoride (NaF)
1 mM sodium orthovanadate (NaV)
1 mM p-nitrophenyl phosphate (PNP)
1 mM Na-ß-glycerophosphate
After 10 mins of lysis I add an equal amount of 20% TCA (4°C) to the solution, vortex and let stand at °4C over night.
Then I follow this protocoll:
1.) Centrifuge the sample(s) at 15.000xg 10 minutes at 4°C.
2.) Discard the supernatant properly.
3.) To the pellet add 800 µl of -20 °C acetone. Vortex the sample back to complete resuspension.
4.) Vortex and let sit at room temp for 5 minutes.
5.) Centrifuge at 15.000xg, 4 °C for 10 minutes.
6.) Discard the supernatant.
7.) Repeat Step 5-7
8.) Discard the acetone and dry the pellet completely for 30 minutes at room temperature.
9.) Resuspend samples in desired buffer.
10.) Let the sample sit for over 1 hour on ice to solubilize all the proteins.
My problems are:
1.) I have a really big pellet after the first step but after the first washing step, it seems smaller. Is this a sign of protein loss?
2.) I can´t resuspend my pellet in aceton for washing! The whole pellet is flying around. After mins of vortexing it falls apart, but in only ~10 pieces. Did I spin to fast/long?
3.) I can´t resuspend my pellet after drying! I tried different times from 5-30 min drying (room temperature or fume hood), so I exclude the possibility of overdrying the pellet. I tried to resuspend in the RIPA-buffer for quantification. Why does my pellet not resuspend?
I will be very thankful for any help!