1.) Using the protein purification techniques covered in class, design a purification scheme to isolate IjkL from the other proteins in solution. It is critical that the protein remains active and correctly folded during the procedure as you would like to conduct biochemical assays on the protein post purification. For each step of the purification, please indicate which procedure has been selected and describe your buffer conditions including buffer, pH, and relative salt concentration. If you need to change buffer conditions, please describe these how you make these changes. At each step, specifically describe which proteins have been maintained in solution and which have been excluded. If appropriate, describe resulting chromatograms, elution procedures, and fractions collected. You can use any of the methods that we have covered in class, but the most efficient, rationally-designed procedures will be given the highest scores. Assume that you know the sequence of the protein and this it is an unmodified wild type sequence.
|
Proteins
|
MW (kDa)
|
pI
|
Percentage of hydrophobic AA
|
Solubility [salt] range Lower bound
|
Solubility [salt] range Upper bound
|
Extinction Coefficient (M-1 cm-1)
|
|
AbcD
|
20
|
7.5
|
32
|
20mM
|
450mM
|
125,500
|
|
EfgH
|
40
|
8.5
|
10
|
20mM
|
300mM
|
80,650
|
|
IjkL
|
45
|
7.5
|
12
|
25mM
|
1M
|
61,200
|
|
MnoP
|
50
|
6.0
|
2
|
30mM
|
1M
|
32,300
|
|
QrsT
|
55
|
4.5
|
5
|
50mM
|
2M
|
30,450
|
|
UvwX
|
75
|
4.5
|
4
|
50mM
|
2M
|
56,750
|
Salting Out: Solubility
Size exclusion chromatography: size exclusion chromatography (MW)
Affinity Chromatography: sequence
Absorbance S
Mass spectrometry: molecular mass
2.) Determine the concentration of your final protein solution if absorbance at 280nm = 10.5
c= A/(∈l)=10.5/(61,200M-1cm-1)(2.80E-5cm))
3.) Using your lecture slides, text, or other sources, propose a method for verifying the identity of your protein (i.e. making sure that your purification scheme worked through outside validation). Describe how thismethod will accurately identify the protein. Assume that you know the sequence of the protein and this it is an unmodified wild type sequence.
Chromatography
Electrophoresis