My sample of interest is highly fibrocartillaginous in nature. I need to extract total protein content from it for which i use RIPA buffer (50mM Tris PH7.4, 150mM Nacl, 0.1% SDS, 0.5% SDC, 1% Triton-X 100) along with 10mM PMSF & Protease inhibitor cocktail.
Since my sample has got more of glycoproteins & proteoglycans, I precipitate it using organic solvents such as methanol-chloroform & acetone. Yet i get these smeary banding pattern as attached in the fig (circled in red)
For membrane proteins and difficult to solubilise material I have developed CTAB-electrophoresis (doi:10.1016/S0003-2697(02)00639-5) as alternative to SDS-PAGE.
The best way to precipitate proteins is that of Wessel & Flügge (doi:10.1016/0003-2697(84)90782-6).
Sharon Miracle Nayagam
With any protein precipitation and then SDS-PAGE Gel you want to make sure you remove as much of the residual solvent as possible. Otherwise it can effect the gel
Thanks for your suggestions Engelbert will try by this method
Shawn Krueger thanks for your comments, i airdry the samples for 5minutes and after complete drying i solubilize it in 1x laemmli buffer yet i get these smeary pattern
can you please tell me can i keep the incubate the samples at 37deg C for complete residual removal??
That isn't necessary. MeOH is quite volatile, residues evaporate at RT within a few min.
Sharon Miracle Nayagam , no you do not want to heat to remove solvent. Let it sit on ice for 30 minutes or RT for 5-10 minutes to make sure it fully evaporates off. If you are still getting smearing, maybe make fresh laemmli buffer