Maleimide/Thiol Assay
September 12th, 2016
Desired Samples to Be Tested
- Maleimide Assay Samples
- Initial liposome maleimide (labelled lipo)
- EGF liposome maleimide (labelled EGFlipo)
- Thiol Assay Samples
- EGF (labelled EGF)
- Thiolated EGF (after dialysis) (labelled TEGF)
Preparation of Cysteine Standard Curve
- Prepared a 2.15 mM stock solution of cysteine in PBS buffer
- Dissolved 13.1 mg of cysteine in 50 mL of PBS
- Weighted on weigh paper and transferred to a 50 mL Falcon tube
- 50 mL of PBS was measure in a graduated cylinder
- Weigh paper wash with 3x 1mL of PBS from graduated cylinder
- 50 mL tube filled half way (~25 mL) with PBS from graduated cylinder and tube was vortexed for 15 sec until dissolved
- Remaining 50 mL PBS added to 50 mL tube and tube was mixed by inverting
- Standard Curve based on Figure 1
- See Table 1 for standard curve dilutions for 8 point standard cysteine curve
- Dissolved 13.1 mg of cysteine in 50 mL of PBS
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
- MEA Working Solution and 4,4-DTDP Stock Solutions prepared by Lily/Tyra
Assay Protocol
- Prepared Total Sulfhydryl (SH Total) reactions
- 3 tubes of each 0.4 mL of Assay Buffer and 0.1 mL of MEA working solution
- Note: Assay buffer solution was not completely thawed when using. (this could effect results, but solution assumed homogeneous; would be better if room temperature)
- Preparation of maleimide assay samples
- Prepared 2 samples at two dilutions (1:5 and 1:10) using the volumes outlined in Table 2
- Note: liposome colour (pink) is still visible at these dilutions
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 |
- Total Sulfhydryl (SH Total) reactions and Maleimide Assay Samples incubated for 20 minutes (4:25 – 4:45 pm)
- Preparation of thiol assay samples
- Prepared 2 samples of EGF (Stock Soln EGF and Thiolated EGF after dialysis) at 1:10 dil, outlined in Table 3
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 |
- Loaded samples into clear flat bottom 96 well plate (200 uL each well) according to Table 4
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
- Samples were read on the plate reader to determine initial absorbance values at a wavelength of 324 nm
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 |
- 10 uL of 4,4-DTDP Stock Solution was added to each well containing sample and standard
- Plate reread at 324 nm wavelength
- Note: no colour change was observed
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 |