All lanes : Anti-IRE1 (phospho S724) antibody (ab104157) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : Jurkat Whole Cell Extract (H2O2 treated)Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Human IRE1 (phospho S724) peptide (ab133861) at 1 µg/mlLane 4 : Jurkat Whole Cell Extract (H2O2 treated) with Human IRE1 (phospho S724) peptide (ab133861) at 1 µg/mlLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ICC/IF image of ab104157 stained Malme-3M cells. The cells were 4% paraformaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab104157, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
![IRE1 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to IRE1 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, Jurkat 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 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab104157.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 110kDa; IRE1](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_3/9905_IRE1-Primary-antibodies-ab104157-3.jpg)
IRE1 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to IRE1 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, Jurkat 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 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab104157.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 110kDa; IRE1