Anti apoptosome anti Nanti protein Xanti caspase based on thé data above where is the WT protein actually cleaved D380D380 and D835D835D835 and D840D840all are cleaved none are cleaved D380 and D840if you compared the size of WT and the D380E mutant before apoptosis on a western blot how would they appear?D380E would be larger they would be indistinguishable WT would be largerhow many amino acids long is the larger band in the lane labeled D380E/D835E **Understanding Site-Directed Mutagenesis and Protein Cleavage in Apoptosis**The 1000 amino acid protein (Protein X) you are studying has several putative caspase cleavage sites. You don’t know how many of them are actually cleaved during apoptosis.Two of them are close enough together that it would be difficult to differentiate the sites just based on the size of the cleavage products. You decide to do some site-directed mutagenesis to figure out what is cleaved and generate mutant constructs for each potential site. You express the proteins in cell culture, induce apoptosis, and do the following Western blot.**Western Blot Diagram Explanation:**The Western blot image shows bands for different mutations of Protein X:- **D380E**- **D835E**- **D840E**- **D380E/D835E**- **D380E/D840E**- **D835E/D840E**- **WT (Wild Type)**Each lane in the blot represents a different mutation or combination of mutations. The presence and size of the bands indicate which cleavage sites are utilized during apoptosis.**Analysis Guide:**- Bands represent cleavage products of Protein X.- Compare patterns between single and combination mutations to identify cleaved sites.- Absence of bands might indicate the prevention of cleavage at specific sites due to mutations.**Question for Further Exploration:**"What antibody did you use here for the Western blot?"- Answer: anti-ApoptosomeThis exercise helps identify crucial sites for caspase cleavage in apoptosis, aiding in understanding protein function and regulation in cell death processes.

Western blotting is a molecular biology technique that is used to detect a specific protein from a complex mixture of protein in a given sample. The proteins are first separated by gel electrophoresis and the results are then transferred to a membrane. The membrane is then incubated with labelled antibodies which are specific to the protein of interest. The unbound antibodies are washed away and only antibodies bound to the protein of interest remain. These bound antibodies are detected by developing a film, only the band of the specific protein will be visible in the film. The thickness of the band tells about the amount of the protein present.Answer 1. Option A is incorrect because anti apoptosome antibody will bind to apoptosome protein only and not to the regions of protein X which is being studied here. Option B is incorrect because anti N antibody will only bind to N protein and will not bind to protein X. Option D is incorrect because anti caspase will bind to the caspase protein and not to the proten X. Option C is correct because anti protein X antibody will bind to the protein X. Answer 2 Option A is incorrect because D380 the banding pattern does not match with the banding pattern of WT protein. Option B is incorrect because D380 and D835 their banding pattern is not the same as the WT protein. Option D is incorrect because D835 and D840 because their banding pattern does not match the WT protein. Option E is incorrect because D840 its banding pattern does not match the WT protein. Option F is incorrect because all sites are not cleaved in the WT protein. Option G is incorrect because none are cleaved does not apply here. Option H is incorrect because D380 and D840 their banding pattern does not match the WT protein. Option C is correct because the bands formed in D835 mutant and WT protein are in the same pattern so, the cleavage site is same.Answer 3. Option A is incorrect because D380E is of the same size as that of WT protein before apoptosis. So, D380E cannot be larger. Option C is incorrect because WT protein is of same size as that of D380 mutant before apoptosis. Option B is correct because the WT and D380E are 1000 aa long proteins and before apoptosis they are not cleaved so they will remain indistinguishable. They are cleaved after apoptosis so they can be distinguished only after apoptosis.Answer 4. The larger band in the lane D380E/D385E is about 900 amino acid long.