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Our Abpromise guarantee covers the use of ab31811 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||1/800. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 57 kDa (predicted molecular weight: 57 kDa).Can be blocked with Human PDI peptide (ab31810).|
|IP||Use a concentration of 5 µg/ml.|
|ICC/IF||Use a concentration of 1 µg/ml.|
PDI was immunoprecipitated using 0.5mg Mouse Pancreas tissue lysate, 5µg of Rabbit polyclonal to 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, Mouse Pancreas tissue lysate 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°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31811.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 57kDa, non specific - as present in control (lane 2); 55kDa: We are confident this was due to slight lane contamination and the band seen in the IP lane is our target of interest; PDI
Image courtesy of Human Protein Atlas
ab31811 staining PDI in Human gall bladder. The paraffin embedded tissue was incubated with ab31811 (1/800 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. ab31811 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.
Further results for this antibody can be found at www.proteinatlas.org
ICC/IF image of ab31811 stained human HEK 293 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab31811, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa and MCF7 cells.