Question

Figure 1. Transient Interaction of Newly Synthesized Proteins with Dnak E. coli spheroplasts were pulse-chase labeled at 30°C, followed by lysis and coimmunoprecipitation (co-IP) of Dnak-polypeptide complexes. (A) SDS-PAGE of Dnak and associated polypeptides. A 15 s pulse of [S]methionine was followed by addition of excess nonradioactive methionine for 15 s, followed by lysis. Total radiolabeled proteins in the soluble fraction (cytosol) of wild-type (MC4100) (lane 1) and Adnak (B81553) spheroplasts (lane 2 were immunoprecipitated with anti-Onak antibody (lanes 3 and 5; equivalent to 15 times the amount loaded in lanes 1 and 2). IP was performed following denaturation with SDS and subsequent dilution (lane 4). IP (as in lanes 3 and 5) was performed, but from a 1:1 mixture of radiolabeled Adnak and unlabeled wild-type spheroplasts (lane 6). Cytosol Anti-Dnak IP -Dnak I II III 1 2 3 4 5 6 wt wt Adnak wt after 1% SDS Adnak labeled Adnak +unlabeled wt

1. Compare Lanes 3 and 4?
   1. What question is being asked?

How tight of a protein-protein association exists?

2. What is the result?

2. Compare lanes 3 and 5?
   1. What question is being asked?

2. What is the result?

I need help with A and B please and can you explain how you got the answers?

Answer 1

A.i) Lanes 3 and 4: Question - What is the effect of SDS treatment on protein protein interaction.

ii) Result - No protein protein interaction is present when a cell is treated with SDS. SDS disrupts interprotein interaction.

B) Lanes 3 and 5: Question - How protein protein interaction is effected in DnaK mutants compared to wild type cells?

Result - Cells mutant for DnaK show no interaction of DnaK and other proteins.