FolA antibody

Cat#: orb813668 (Validation Data)

Antibody Nam e: Rabbit anti -Escherichia coli (strain K12) folA Polyclonal antibody
1. Uniprot N o.: P0ABQ4
2. Antigen Preparation
3. Antigen Identification by S D S -PAG E:

AA Sequence: 1-159aa
Vector:pET21a
Predicted MW: 18.8kDa
Observed MW: 20kDa
Conclusion : The observed molecular weight of antigen by SDS-PAGE is larger than the predicted value, the
difference is related with the protein molecular structure. The pI value of fusion protein is 6.42.The essential
reason is that acid protein itself is negatively charged, and it will bind less SDS, so the migration will be slower
under the action of electric field. Overall, the test result is right.
4. Immunization of Rabbits
5. Determination of anti-serum titer by indirect E LIS A
1) Coating antigen in 2ug/ml, overnight under 4℃.
2) Blocking with 5% milk (non-fat dry milk dissolved in PBST) for 2 hours under 37℃.
3) Dilute the anti-serum proportionally at 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000. Add
100ul anti-serum per well, and set pre-immune serum as negative control.
4) Incubate primary antibody with sample diluent under 37℃ for 1 hour.
5) Dilute secondary antibody at 1:50000 with 5% milk (non-fat dry milk dissolved in PBST), incubate for 30
minutes under 37℃.
6) Add TMB, then incubate for 15 minutes under 37℃.
7) Add Stop Solution, react with 15 minutes, then read the optical density of each well using a microplate reader
set to 450nm.

6. Antibody Purity Determination by S D S -PAG E

Purity: 98% by SDS-PAGE

Serum # DK-1179

6. Western Blot Detection with Purified Antibody

1) Sample loading: 10ul per well (~20ug); Electrophoresis: 12% gel, 80V ~30min, 120V ~60min.
2) Semi-dry transfer: 60mA, 80min (0.45PVDF, Millipore).
3) Blocking: 5% milk (non-fat dry milk dissolved in PBST) for 2 hours under 25℃.
4) Primary antibody incubation: Dilute primary antibody at 1:2000 with 5% milk (non-fat dry milk dissolved in
PBST), incubate for 1 hour under 25℃.
5) Washing: PBST, 4 times*10min.
6) Secondary antibody incubation: Dilute secondary antibody at 1:50000 with 5% milk (non-fat dry milk dissolved
in PBST), incubate for 1 hour under 25℃.
7) Washing: PBST, 4 times*10min.
8) ECL chemiluminescence and X-ray imaging.

Western Blot with Antigen

Conclusion
The antigen is Recombinant Escherichia coli (strain K12) folA protein (1-159aa) and the antigen was expressed in
E. coli system, linked the vector of pET21a.
The predicted molecular weight of antigen should be 18.8kDa.
The observed molecular weight of antigen by SDS-PAGE is about 20kDa.
The observed molecular weight of antigen by Western Blot is 20kDa.
The observed molecular weight of antigen by SDS-PAGE is larger than the predicted value, the reason has been
explained before. The observed molecular weight of antigen by Western Blot is equal to the SDS-PAGE result. In
conclusion, we can confirm is specific to recognize antigen, the validation results of
antigen and antibody are correct, and the quality control was passed.