Anti-ERp57 antibody (ab10287)

All lanes : Anti-ERp57 antibody (ab10287) at 1/1000 dilution

Lane 1 : Mouse embryonic cells
Lane 2 : Calreticulin deficient mouse embryomic cells

Predicted band size: 61 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?

Western blot using ab10287 at 1/1000.

Lane 1 – Mouse Embryonic Fibroblasts.
Lane 2 – Calreticulin deficient Mouse Embryonic Fibroblasts.

Double staining of mouse embryonic fibroblasts (top) and calreticulin deficient fibroblasts (bottom) with rabbit-anti-ERp57 antibodies (1:100;  upper and lower left panel) and FITC-ConA (upper and lower right panel).

ab10287 at 1/100 staining human HeLa cells by ICC/IF. The cells were premeabilized with digitonin before being blocked and then stained with the antibody for 1 hour. An Alexa-Fluor ® conjugated goat anti rabbit antibody was used as the secondary.

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ab10287 (1/2000) staining ERp57 in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic staining.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

ERp57 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to ERp57 (ab10287) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab10287.
Secondary: Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 61kDa: ERp57