BIOC12 Lec 4 slides 1-9

Question 1

what is the purpose of western blotting

Answer 1

Done to confirm the presence of a specific
protein in a protein population
* Separate proteins on SDS-PAGE, transfer
to a membrane and probe with an
antibody specific to the antigen(protein) of
interest

Question 2

what are the 6 steps of western blotting

Answer 2

slide 4

Question 3

what is the purpose for Amino acid hydrolysate

Answer 3

to determine protein composition

Question 4

how does Amino acid hydrolysate work

Answer 4

. First, the peptide bonds of the protein are cleaved by acid hydrolysis, typically using 6 M HCl

Next, the hydrolyzed mixture, or hydrolysate, is subjected to a chromatographic procedure in which each of the amino acids is separated and quantitated, a process called amino acid analysis.

Question 5

One method of amino acid analysis involves treatment of the protein hydrolysate with phenylisothiocyanate (PITC) at pH 9.0 to generate phenylthiocarbamoyl (PTC)–amino acid derivatives (Figure 3.16)

Answer 5

rememeber

Question 6

how does treatment of the protein hydrolysate with phenylisothiocyanate (PITC) work

Answer 6

page 4 of chapter 4 key notes

Question 7

what is the purpose of Chromatograph from HPLC
separation of the PTC-amino acids in
the column

Answer 7

These represent the amino acids in the original
protein: amount is equal to the area under the peak
*limitations:
B and Z peaks: cannot measure glutamate or aspartate
-destroys some side chains such as tryptophan
and does not accurately determine S, T, Y