I need help, please. mportant to known the protein concentration when performing
ID: 716760 • Letter: I
Question
I need help, please.
mportant to known the protein concentration when performing enzyme aeti studies? 12. A mixture of cytochrome c (human), myoglobin (equine heart), serum albumin (Chuman) and apolioprotein B (human) were subjected to size-exelusion chromatography a. Which protein is likely to be retained the longest (i.e , will clute last) on the column? b. Which protein would show up as the band at the top of an SDS-PAGE analysis? 13. Ou tline the individual steps required to sequence a protein. What must be done to apply the sequence before larger proteins can be sequenced? 14. Assume you have isolated a relatively abundant protein, and you want to obtain the amino acid sequence. You added dithiothreitol to the sample then did SDS-PAGE and found two bands A at 2.9 and B at 3.9 kD,. You then cleaved the protein with chymotrypsin and found the following fragments: Band A CN Band B F NLQNY GIVEQCCHKRCSEY DPTKM ACGVRGF RTTGHLCGKDLVNALY You then cleaved with Staphylococcus aureus V8 protease and found the following Band BPTKM Band A GIVE RTTGHLCGKD YNLONYCN LVNALYIACGVRGFFYD QCCHKRCSE What is the primary amino acid sequence of the protein?Explanation / Answer
Answer 12:
Size exclusion chromatography is a technique used to separate molecules by their size. A diluted sample is passed through a column which is filled with a porous and inert material. As the sample is flowing down, small molecules penetrate the pores of the filling material while molecules that are larger than the pores are unable tu diffuse through them, so they flow faster through the column. Therefore, larger molecules elute first.
Molecular weight can give us an idea of the difference in size between the proteins. Let's order the proteins from smaller to largest molecular weight:
Cytochrome C (human) < Myoglobine (equine heart) <Serum Albumin (human) < Apolioprotein B (human)
So, Cytochrome C (human) would be the last protein to elute.
As for the SDS PAGE analysis, it is a technique that separate proteins by their mobility due to their molecular sizes. When SDS (sodium dodecyl sulfate) is added to the sample, it unfolds the protein by forming linear SDS - protein complexes, and also coats it with an uniform negative charge, making its charge proportional to its molecular weight. Now, when the sample is passed through a matrix and an electric field is applied, the mobility of the proteins will depend only on its molecular weight. Small molecules will migrate faster than large molecules. Therefore, Apolioprotein B will show up as the band at the top of the analysis.