In order to check the interaction of StrepDARPidin with the EpCAM on A549 Lung Carcinoma cells we planned an ELISA-Like assay again. Yesterday row F 1-12 of a black Fluotrac600 96-well plate was coated with 100000 cells A549/ well (counted with Neubauer-Chamber) overnight at 37°C. 100000 cells of 3T3 fibroblasts were used as negative control and as well used to coat well G6.
We intended to incubate them with different concentrations of StrepDARPidin and then target the N-terminal His-Tag with and Anti-His antibody Alexa488 conjugated (1:50 endvolumen).
In wells H1-6 gel filtration purified StrepDARPidin with 87 µM was tested, in well H 7-12 Ni-NTA purified StrepDARPidin with 280 µM.
Following concentrations of StrepDARPidin dissolved in PBS (pH 7,4) +2,5 % glycerin were used:
Content |
Well
Col |
Well Row |
raw data (485, 520) |
Purification |
StrepDARPidin concentration [M] |
A549 |
1 |
F |
5782 |
GeFi |
50 µM |
A549 |
2 |
F |
4912 |
GeFi |
25 µM |
A549 |
3 |
F |
3903 |
GeFi |
2,5 µM |
A549 |
4 |
F |
3513 |
GeFi |
250 nM |
A549 |
5 |
F |
3613 |
GeFi |
25 nM |
A549 |
6 |
F |
3211 |
GeFi |
2,5 nM |
A549 |
7 |
F |
7340 |
Ni-NTA |
50 µM |
A549 |
8 |
F |
5329 |
Ni-NTA |
25 µM |
A549 |
9 |
F |
5846 |
Ni-NTA |
2,5 µM |
A549 |
10 |
F |
5718 |
Ni-NTA |
250 nM |
A549 |
11 |
F |
3852 |
Ni-NTA |
25 nM |
A549 |
12 |
F |
6828 |
Ni-NTA |
2,5 nM |
Empty well |
5 |
G |
5699 |
- |
- |
negative control 3T3 fibroblasts |
6 |
G |
9983 |
- |
- |
Blank |
7 |
G |
1760 |
- |
- |
positive control AB |
8 |
G |
60182 |
- |
- |
The 96-well plate was taken out of the incubator and the medium was carefully discarded. 100 µL of the dilutions were transferred into the wells and incubated for 45 min. at room temperature under the Flow hood.
An Anti-His-Alexa488 conjugate (1 mg/mL stock) with 5 µL concentrate was dissolved in 40 µL PBS. 1 µL of the antibody was added after washing to each well and incubated for 20 min at room temperature in the dark.
As a negative control t-cells clones were used and incubated the same way with 1 µM StrepDARPidin and 1 µL antibody (Well G8). Well G5 was left empty, well G7 was filled with 100 µL PBS+Glycerin and G4 with PBS+2,5 % glycerin with 1 µL Antibody. As negative Bacillus Subtilis WT culture was used which was already lysed causing the huge signal. A different EpCAM-negative cell line has to be used.
After 20 min incubation the wells were washed carefully with 100 µL PBS to wash out unbound elements.
The plate was measured in the ELISA reader:
We are able to see that the fluorescence signal decreases with increasing dilution. Unfortunately the negative control was very high as well. The t-cells were incubated in Eppis because of their non-adherent features. They were incubated with StrepDARPidin and Antibody before. The supernatant was discarded after centrifugation at 1500x g but the cells did not stay at the pellet’s position so that it was not possible to remove the whole preincubated material which might be left when measuring the control.
Additionally it is noticeable that there is the same trend for both purification methods. The lower signal from NTA-purified StrepDARPidin can be explained by the fact that the concentration was higher but the purity also lower so that even less StrepDARPidin was found in the protein aliquods. As negative Bacillus Subtilis WT culture was used which was already lysed causing the huge signal. A different EpCAM-negative cell line has to be used.
For the next measurement row C of the plate was coated with 100000 A549 cells & Caco-2 cells per well overnight and the same amount of 3T3 fibroblasts as negative control.