A Western blot was performed to validate the STJ92925 Anti-Endoplasmin Antibody using HeLa cell extracts. A band was observed around 92kD on the membrane, indicating the antibody recognized endoplasmin in the HeLa extracts as expected. The validation involved lysing HeLa cells, running protein samples on a 10% polyacrylamide gel, transferring to a PVDF membrane, blocking with milk, probing with the primary and secondary antibodies at specified dilutions, washing, and visualizing with ECL.

1. Result
“Specific band in HeLa extracts.” - A. Diaz, Centro Nacional de Biotecnologia CNB CSIC.
Cell Line: HeLa
Method of validation: Western Blot
Primary Antibody:
STJ92925 Anti-Endoplasmin Anti-
body
Secondary Antibody: HRP goat anti-rabbit
Dilution ratio: 1:1000
Protocol
Treatment of materials: When cells were cultured to 80% plating density, lysis buffer with triton X-100 were added, soluble and insoluble frac-
tions were separated by centrifugation and sample buffer was added and then boiled.
Gel electrophoresis: 10% polyacrylamide gel, constant voltage of 160V for 60 minutes.
Transfer: 0.45 µm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 minutes.
Blocking: 1X TBST with 5% skimmed milk powder was used and left for 1 hour.
Primary antibody probing: Primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder at 4˚C and left overnight.
Membrane wash: Membrane was washed 3 times with 1X TBST.
Secondary antibody probing: secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with 1:10000 dilution ratio,
for 1 hour.
Membrane wash: Membrane was washed 3 times with 1X TBST.
Visualization: : ECL visualization for 5 - 10 minutes.
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STJ92925 Anti-Endoplasmin Antibody
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Gel electrophoresis information: 10% polyacrylamide gel, constant voltage of 160V for 60 minutes.
Transfer information: 0.45 µm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 minutes.
No. Antigen
Loading
amount
Primary anti-
body
Primary
antibody
dilution ratio
Secondary
antibody dilu-
tion ratio
Target band KD Visualization time
1 HeLa Extract 10µg
STJ92925 Anti-
Endoplasmin
Antibody
1:1000 1:10000 ~92KD N/A