Synthesis of EGF


Maleimide/Thiol Assay

September 12th, 2016

Desired Samples to Be Tested

Preparation of Cysteine Standard Curve

Figure 1: Cysteine Standard Curve


Table 1: Standard Curve Dilutions

Tube Standard (uL) PBS (uL) Final Concentration (mM)
1 0 500 0
2 100 of 3 400 0.050
3 750 of 4 750 0.125
4 750 of 5 750 0.250
5 750 of 6 750 0.500
6 1000 of 7 500 1.000
7 1047 of 2.15 mM Std. 453 1.500
8 500 of 2.15 mM Std. 0 2.150

Stock Solutions

Assay Protocol

Table 2: Maleimide Assay Sample Prep

Sample Sample Volume (uL) Assay Buffer (uL) MEA (uL) Sample Concentration
Lipo1 100 300 100 1:5 dil
Lipo2 50 350 100 1:10 dil
EGFLipo1 100 300 100 1:5 dil
EGFLipo2 50 350 100 1:10 dil


Table 3: Thiol Assay Sample Prep

Sample Sample Volume (uL) Assay Buffer (uL) Sample Concentration
EGF 50 450 1:10 dil
T EGF 50 450 1:10 dil

Table 4: 96 Well Plate Configuration

  1 2 3 4 5 6 7 8 9 10 11 12
A 0 0 x SHTotal1 SHTotal1 x EGF EGF x x x x
B 0.050 0.050 x SHTotal2 SHTotal2 x T EGF T EGF x x x x
C 0.125 0.125 x SHTotal3 SHTotal3 x x x x x x x
D 0.250 0.250 x x x x x x x x x x
E 0.500 0.500 x Lipo1 Lipo1 x x x x x x x
F 1.000 1.000 x Lipo2 Lipo2 x x x x x x x
G 1.500 1.500 x EGFLipo1 EGFLipo1 x x x x x x x
H 2.150 2.150 x EGFLipo2 EGFLipo2 x x x x x x x

columns 1 and 2: standards
SHTotal1, SHTotal2, Lipo1, Lipo2, EGFLipo1, EGFLipo2: maleimide samples
EGF, T EGF: thiol samples
x: blank

Table 5: Initial Read of Plate at 324 nm

Value 1 2 3 4 5 6 7 8 9 10 11 12
A 0.166573 0.165342   0.165488 0.159803   0.162751 0.166633        
B 0.167365 0.163468   0.160715 0.157624   0.161829 0.162791        
C 0.167854 0.164503   0.162529 0.158517              
D 0.166595 0.169136                    
E 0.167545 0.183346   1.87872 1.90161              
F 0.167228 0.169563   1.13511 1.11714              
G 0.174248 0.16986   1.06884 1.09707              
H 0.180233 0.169048   0.642909 0.643084              

Table 6: Read after Addition of 4,4-DTDP

Value 1 2 3 4 5 6 7 8 9 10 11 12
A 0.575088 0.516103   1.07039 1.04125   0.532653 0.526909        
B 0.56686 0.534722   0.86236 1.15278   0.530382 0.526023        
C 0.577889 0.567224   0.908712 1.13684              
D 0.568944 0.520478                    
E 0.566859 0.554068   2.5377 2.55986              
F 0.573003 0.54943   1.76687 1.83494              
G 0.574038 0.557013   1.73797 1.78014              
H 0.527355 0.564607   1.3762 1.26635              

Protein Assays

Synthesis of APOE