I isolated protein from Huh-7 cells fraction using lysis buffer (0.5% SDS, 100 mM DTT in 20 mM Tris). Lysed precipitated by 20% TCA and washed several times with Acetone. Finally resuspended in rehydration buffer (8M Urea, 2% CHAPS, 2M Thiourea, 2% ASB, 5% IPG buffer; 65mM DTT). The pellet is very hard to dissolve! Although the same pellet dissolves promptly in the above mentioned Lysis buffer.
do not air dry pellet to long. When pellet is dry for long time it does not dissolve in rehydration buffer
do not air dry pellet to long. When pellet is dry for long time it does not dissolve in rehydration buffer
Agree with Ravinder, don't dry pellet for long. Once washed with Acetone (I use methanol) leave it open at room temperature once you don't see any moisture (~5mins) and then resuspend in desired buffer. If its still not soluble then used water bath sonicate (avoid vortex to prevent frothing).
If the downstream process is mass spectrometry them try dissolving in 50:50:0.1 Water:Acetoitrile:TFA.
If the protein content is too low substitute TFA with acetic acid or formic acid.
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