Five Tips to Successful Western Blot of phospho-IRE1 alpha (Ser724)

Phospho-IRE1 alpha (Ser724) antibodies will detect IRE1 alpha protein only when it is phosphorylated at the Serine-724 amino acid position. If IRE1 alpha is not phosphorylated (activated) in the samples being tested, phosphorylation specific IRE1 alpha antibodies would not generate a signal. Therefore, it is recommended to use a positive control from cells with high amounts of ER stress/UPR activation. Our in-house testing for phospho-IRE1 alpha (Ser724) expression with Novus’ phospho-IRE1 alpha (Ser724) antibody (NB100-2323), has demonstrated that Min6 cells exposed to increasing concentrations of glucose (up to 20nM for 3 hours) or HeLa cells exposed to Dithiothreitol/DTT (10 mM for 1 hour) serve as great options for positive controls in phospho-IRE1 alpha expression analysis through Western blot.

pSer724 IRE1 alpha [NB100-2323] WB detection of pSer724 IRE1 alpha Min6 cells treated with increasing concentrations of glucose for 3 hours prior to lysate preparation.		Total IRE1 alpha [NB100-2324] WB detection of total IRE1 alpha protein in lysates from Min6 cells which were transfected with control GFP-siRNA or IRE1 alpha-siRNA.

2. Protein Separation

An effective separation of the sample proteins in SDS-PAGE gel is highly critical for Western blot detection, especially in the case of high molecular weight proteins. IRE1 alpha’s molecular weight is ~110 kDa, so a lower percentage gel will help achieve better separation. Specifically, we recommend using a 6%, 8%, or a gradient gel for Western blot analysis of phospho-IRE1 alpha (Ser724) or total IRE1 alpha.

Like other high molecular weight proteins, the transfer of phospho-IRE1 alpha also requires optimization. An extended transfer at 4˚C is recommended when transferring proteins from gel to the membrane. For enhancing the transfer process, the transfer buffer may be supplemented with SDS (not more than 0.1%). After the transfer step is completed, we recommend confirming a successful protein transfer, particularly in the high molecular weight range (90-130 kDa), by staining the membrane with Amido black or Ponceau S stains. 

When working with phosphorylated proteins in Western blot, one of the most common issues is high background in the signal detection step. To probe the expression of phosphorylated proteins, including phospho-IRE1 alpha, we recommend 5% BSA in TBST as a blocking buffer. A milk based blocking buffer should be avoided because the milk contains phospho-proteins which may increase background signal. Additionally, 1% BSA in TBST should be used as an antibody diluent buffer when incubating the membranes with primary and secondary antibodies. If high background signal is not resolved by using BSA based blocking buffer, a section of the membrane may be cut and incubated with secondary antibody only (no primary antibody) to see if the source of the background is the secondary itself.

To accurately determine changes in the expression of phospho-IRE1 alpha (Ser724), it is important to analyze expression levels of both phospho- and total-IRE1 alpha. Modulations in phospho-IRE1 alpha (Ser724) expression should be compared to total IRE1 alpha expression to ensure changes in the abundance of the activated form are real, and are not the result of an increase in the total pool of IRE1 alpha. An ideal method to probe total protein expression is to strip the membrane after phospho-IRE1 alpha (Ser724) detection and re-probe it with an antibody against total IRE1 alpha (such as Novus’ NB100-2324 and NB110-59971). We also recommend using a loading control with mid to high molecular weight (such as Tubulin) to determine if the observed changes in IRE1 alpha are real or just the result of an unequal loading.

Also read UPR and ER Stress FAQs for scientific technical answers to the most frequently asked questions on ER stress, UPR signaling, troubleshooting tips on UPR markers (pSer724 IRE1 alpha, cleaved ATF6, pPERK, GRP78 etc.),  IRE1 alpha positive control, inhibitors/inducers and more.