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IPTG induction of GST or GST-YAR1
Your protein samples from week 10 were loaded onto 12.5% polyacrylamide gels for SDS-PAGE. The gels were run at 20 mA of current for approximately 1 hour, or until the dye front neared the bottom of the gel. They were then removed from the apparatus and stained for 10 minutes with a blue dye (Coomassie brilliant blue), which binds tightly and nonspecifically to proteins. They were then soaked overnight in a destain solution (10% acetic acid), which removes background staining so that the protein bands remain stained but the rest of the polyacrylamide gel returns to clear. Lastly, they were dried overnight between sheets of cellulose so that they could be stored indefinitely. The dried-down gels were scanned with an Epson 1200 color scanner, and the scanned files were converted to JPEG format with Adobe Photoshop 5.5.
10µl of each protein sample was loaded per well, except for the lysate samples, for which 20µl was loaded. This should represent approximately 0.1 "OD units" of cells per lane--the lysate samples were 2X more dilute than the others, so twice as much was loaded.
The order of experimental lanes for each set of samples (reading from left to right) was uninduced, 1 hour, 2 hours, and then lysate. (The four lanes in each figure that have lots of bands in them are the experimental lanes--the other lane is the ladder.)
Bio-Rad "broad range" molecular weight standards were used in 1-2 lanes per gel, and are included in each scan. The sizes of the bands that are visible in these gels are (in decreasing order): 200 kDa, 116 kDa, 97 kDa, 66 kDa, 45 kDa, 31 kDa, and 21.5 kDa. The largest bands are at the top of the gel, and the smallest at the bottom.
Click on your group's initials to see the scan of your gel. You should print out this scan and label it appropriately for your lab report. See your lab handout for details.
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