Question

I need help with all parts please.

Cytosol Anti-Dnak IP Anti-Dnak IP -Dnak -Dnak IIIIIIII Proteins bound to Dnak (Arb. Units) 1 2 3 4 5 6 0 2 8 10 4 6 Time (min) i Time (min) Adnak wt Adnak wt after 1% SDS labeled Adnak +unlabeled wt 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 [35S]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 (BB1553) spheroplasts (lane 2) were immunoprecipitated with anti-Dnak 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 (ane 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). (B) SDS-PAGE of DnaK and associated polypeptides labeled and immunoprecipitated as in (A), with excess methionine added at time 0. (C) Quantification of the data in (B).

1. Compare Lanes 1 and 2?
   1. What question is being asked?

2. What is the results

B.) Compare Lanes 3 and 4?

                     How tight of a protein-protein association exists?

Answer 1

A. Lanes 1 and 2: Question - Is there any difference between total protein content of wild type cells and dnaK spheroplasts?

Result - There is no visible difference between the total protein present (no IP) in the wild type cells amd dnaK spheroplasts as both show a comparable protein smear in the lanes.

B. Lanes 3 and 4: Lane 3 has IP done with wild type cells while lane 4 has IP done with SDS treated cells. SDS treatment abolishes any protein protein interaction in the cell, which is why no protein is seen to interacting with dnaK, compared to wt cells.