Thank you!! As you know, protein kinase A phosphorylates amino acid residues on

Question

Thank you!!

As you know, protein kinase A phosphorylates amino acid residues on target proteins that are part of a specific peptide region. An antibody raised against the phosphorylated form of this peptide (phospho- PKA substrate antibody) can be used to identify and study PKA-phosphorylated target proteins Figure 1 shows the results of an experiment in which cell extracts were in vitro phosphorylated by protein kinase A, in the absence (Sample 3) or presence (Sample 4) of a PKA inhibitor (PKI). For comparison, two other protein kinases, extracellular signal-regulated kinase 2 (ERK2; sample 6) and a CyclinA/Cdkl complex (sample 8) were also tested with the same cell extract. Mixtures without cell extracts (Sample 2, 5, and 7) served as control samples. After incubation samples were subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis using the phospho-PKA substrate antibody

Explanation / Answer

Ans) The phospho-PKA substrate antibody binds specifically to the phosphorylated peptide by PKA enzyme. This enables to study about the target proteins phosphorylated by PKA. In the present study, other kinase ERK2 and Cyclin/CdK is used to analyse whether these kinases too phosphorylate the substrate in the manner similar to that of the PKA by western blot in which phospho-PKA substrate antibody is used.

Lane 1: Control

Lane 2: PKA + ERE2 + Cyclin/CdK

Lane 3: Cell extract + PKA

Lane 4: Cell extract + PKA + PKI

Lane 5:PKA + ERE2 + Cyclin/CdK

Lane 6: Cell Extract + ERE2

Lane 7: PKA + ERE2 + Cyclin/CdK

Lane 8: Cell Extract + Cyclin/Cdk

2.5) A) The given blot has clear bands at lane 3 (total length of the lane) suggesting that it has phosphorylated the target proteins in the cell extract.

2.6) B) On comparing lane 3 and lane 6, the band has not appeared similar (no thick bands) to that of treated with PKA. Thus, ERE2 has phosphorylated different target peptides.

2.7) C) Though there is a difference in the phosphorylation of target peptides, it is impossible to conclude that ERE2 phosphorylate the same protein which the PKA has phosphorylated or not.

2.8) A) This is true because there is no thick band seen in the lane 4, suggesting the PKI block the PKA and make it unavailable for the antibody to bind.

2.9) C) The antibody to bind is specific for PKA phosphorylated peptide targets and this experiment does not give any result about the acitivity of Cyclin/Cdk. Moreover, the enzyme might have phosphorylated other peptides which can't be determined by PKA specific antibody

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