Finding the # of protein bands Circling the phosphorylated forms Cell Culture Gr

Question

Finding the # of protein bands

Circling the phosphorylated forms

Cell Culture Growth

11. Phosphorylation of the mitochondrial protein Bax leads to cell death. You have a MAb that recognizes the phosphorylated form of Bax and a polyclonal anti-serum that recognizes both the phosphorylated and non-phosphorylated forms of Box. Crude cell extracts from cultures grown at 25 degree C and 30C were run on an SDS-PAGE and transferred to a nylon membrane. The membrane was stained/destained with Ponceau S, blocked with BSA, probed with the polyclonal anti-serum, and detected with a secondary antibody. The nylon membrane was then stripped, re-probed with the MAb, and detected with a secondary antibody. The composite data is shown below. (2pts) How many protein bands would you expect to see using the Ponceau S procedure? (1pt) On the blot probed with Bax/Bax-P anti-serum, circle the phosphorylated form(s) of Bax. (1pt) Which cell culture growth temperature favors phosphorylation of Bax? (2pts) At which cell culture growth temperature would you expect to find more living cells? (3pts) Is there non-phosphorylated Bax present on the far right lane (yes/no)? How do you know?

Explanation / Answer

1. Ponceau S will stain all the proteins present in the crude cell culture, giving a general view of the proteins present in the extract.

2. The thick band is of phosphorylated form of bax

3. 30oC

4. 30oC

5. No. The MAb recognises only phosphorylated form of Bax

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