I want to run an SDS PAGE of rice crude proteins. I used a grinding method with liquid nitrogen, but was unable to make a good sample that I could use for SDS PAGE. Is there any other suggested method that can be effective?
actually i grind my sample in liquid nitrogen then add protein loading dye and after boiling load it into SDS PAGE..but i am not getting proper bands..so i am doubtful about the sample..
I used the eppendorf tubes and micropestles to grind about 1 sq cm piece of rice leaf tissue in liq N2, and allowed the ground tissue to thaw completely on ice. Once thawed, I added about 300 ul of the below lysis buffer with proteinase inhibitors, and incubated samples on a rocker for 1 hr at 4c. Spin at high speed at 4c and collect the supernatant. You can quantify the protein content and store the lysate at -80. After quantification aliquot the required concentration of protein and dilute it with the required volume of 4x sample buffer, boil and then run them.
Hope this helps.
Lysis buffer: 0.1 M Tris, pH 7.5; 10% sucrose; 5 mM EDTA, pH 8.0; 0.2% EGTA; 0.3% bME.
Make sure you add reducing agent. Rice proteins will aggregate when heated.
I agree with Wilfredo, make sure that there is a reducing agent in your sample buffer and if bME doesnt work then use DTT instead. Your sample after mixing with the sample buffer containing reducing agent should still be blue in color, if it changes to any other color then your samples wont resolve properly on the gel.
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