Question

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).
I nedd help with all parts please.

Answer 1

1. If we place radiolabeled methionine for a short duration (15 seconds) and non radiolabeled for 30 seconds followed by immunoprecipitation with a specific antibody determines the presence or absence of that particular protein in the sample at different time intervals.

2. Lane 1 and 2 are the radiolabeled fractions of solubulized proteins in the cytosol of wild type (lane 1) and dnaK proteins of spheroplasts ( lane 2). These two lanes includes total radiolabelled proteins of the two samples. The presence of these two proteins in these two samples looks no visible Dnak proteins or very less.

3. Immunoprecipitation is a technique to separate a specific protein of our choice using s antibody against to it ( here it is DnaK). This procedure will quantify the presence of that particular protein even if it is present in less amount.

4. Lane 1 is the radiolabeled total proteins of the soluble fraction of cytosol of wild sample in which there is no visible presence of DnaK is present but when it is immunoprecipitated with anti DnaK antibody there is a specific band of DnaK is present (lane 3) which shows that in the total sample the protein presence is less but when quantified, alone protein was precipitated showing its presence.