I have expressed one viral gene in Pischia pastoris. the expressed recombinant protein is secreted in the supernatant. I have concentratexd the supernatant using acetone and TCA method and run the samples on 12.5% SDS-PAGE. I have done it thrice but every time I am getting a smear rather than resolved protein bands besides markers also twisting. I am afraid is it because of acetone.
Hi,
you get a smear probably because of glycosilation!
Pichia is able to glycosilate protein but not homogeneously, so you have different amount of sugar linked to you protein.
To verify if your smear is due to glycosilation you can try to cut the sugar attached to the protein with an enzyme like this one https://www.neb.com/products/p0704-pngase-f (PNGase F), and run a gel. If you cut all the enzyme you might get a band instead of a smear.
Hi
The TCA can give you quite nasty smears. Before you run you samples after precipitating them you may want to do an acetone wash. Resuspend the pellet in ice cold 80% Acetone (sometimes I supplement with DTT to start reducing the cysteine bonds which can lead to protein aggregation resulting in smears on your gel), spin this to collect the pellet and discard supernatant. Do this 2 to 3 times. Air dry your pellet....do not air dry it for too long as it will be difficult to bring the pellet into solution again. Once you have done this, you can do one of the three:
1) Resuspend the pellet in 2X Laemli buffer, boiled and load
2) Re-solubilise you pellet in Urea lysis buffer (7 M Urea,2 M Thiourea, 4% CHAPS) (I would advise that you also add DTT in this to keep your protein reduced, about 10 to 50 mM). Vortex this 1 to 3 hours to solubilise your pellet. Spin @ 10 000 rpm. Aliquot from the supernatant mix laemli buffer to 1X final concentration, boil and load.
3) Solubilise the pellet in PBS buffer (You can supplement with DTT) add laemli to a final concentration of 1X, boil and load
All the best
Thanks Lusisizwe Kwezi...could you please provide the concentration of DTT to be used..
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