Endogenous IRE1 protein levels are predicted to be very low because it is a stress sensor that mediates alternative splicing of a transcription factor transcript, and there is little evidence that IRE1 protein levels change much during the unfolded protein response. So it will always be hard to detect by standard western blots and you may have to partially fractionate your cells to enrich for ER/nuclear enveloppe membranes so you can load more total protein on gels. To get the detection to work routinely so you can study phosphorylation or any other post-translational modification to IRE1 (such as a modified subcellular localisation and potentially increased turnover), you must first establish the detection limit of your antibody. This is easiest done by artificially overexpressing IRE1 in a suitable cell line and comparing an extract of wild type cells with extracts from the transfected or transformed cells. The latter should either show an additional band around 110 kDa or an intensified band at that position when it is already present in the wild type cells. You may also see more of the small band you are detecting at the moment (this would indicate that it is an IRE1 degradation intermediate). Once you have establish the detection limit and you are sure that the bands you are seeing correlate with the overexpression of IRE1, you can start working out what method to use to detect the endogenous IRE1 levels (that may be below the detection limit for a crude total extract).

Endogenous IRE1 protein levels are predicted to be very low because it is a stress sensor that mediates alternative splicing of a transcription factor transcript, and there is little evidence that IRE1 protein levels change much during the unfolded protein response. So it will always be hard to detect by standard western blots and you may have to partially fractionate your cells to enrich for ER/nuclear enveloppe membranes so you can load more total protein on gels. To get the detection to work routinely so you can study phosphorylation or any other post-translational modification to IRE1 (such as a modified subcellular localisation and potentially increased turnover), you must first establish the detection limit of your antibody. This is easiest done by artificially overexpressing IRE1 in a suitable cell line and comparing an extract of wild type cells with extracts from the transfected or transformed cells. The latter should either show an additional band around 110 kDa or an intensified band at that position when it is already present in the wild type cells. You may also see more of the small band you are detecting at the moment (this would indicate that it is an IRE1 degradation intermediate). Once you have establish the detection limit and you are sure that the bands you are seeing correlate with the overexpression of IRE1, you can start working out what method to use to detect the endogenous IRE1 levels (that may be below the detection limit for a crude total extract).

Thank you Jürgen Denecke, very helpful advices !!! I'll try first to find the detection limit of my antibody and after that I'll try the other suggestions.

I'm getting back to work now

Thank you again

Have a nice week

Great feedback so far and I totally agree with the suggestion of a positive control in the assay for getting the issue resolved. However, because the antibody under consideration resulted in a major band at 20-28 kDa position, there is a chance that the Lot of the antibody has gone bad or the antibody in the Lot being tested is binding to some other low molecular weight protein (hence not specific for pS724-IRE1 Alpha).

As far as the detection limits are concerned, several companies offer antibodies to phospho-IRE1 Alpha with validation data from commonly used cell lines exposed to ER stress conditions. We from Novus provide antibodies for Total as well as phospho-Ser724 IRE1 Alpha with over 100 citations in peer reviewed journals (http://www.novusbio.com/ire1-alpha.html). I would recommend reviewing some of those publications to see how other researchers have used phospho-IRE1 Alpha antibodies successfully in similar experimental model.

Since phospho-S724 IRE1 Alpha antibodies will detect IRE1 Alpha protein when phosphorylated at Serine-724 amino acid position, using a positive control from cells with considerable amount of ER Stress would be a more ideal control compared to IRE1 Alpha over-expression lysate. In the datasheet of our NBP2-50067, we have shown an enhanced levels of pS724-IRE1 Alpha in total cell lysates from Min6 cell line exposed to increasing concentrations of glucose (up to 20nM for 3 hours). Glucose is probably the least expensive option, but other compounds which induces phospho-IRE1 Alpha levels are tunicamycin and Calyculin A.

For Top 5 Tips to Successful Western Blot of phospho-IRE1 alpha (Ser724), read https://www.novusbio.com/node/12415896 

The antibody offered by Abcam is identical to the one offered by Novus Biologicals. Look at the blots on the product sheets. No need to purchase it again.

I tested the anti-phospho-IRE1a by myself. Phospho-IRE1a specific band (110kDa) is very very faint (40 ug protein ECL based detection). Many unspecific bands display stronger signal intensities. I would not be surprised at all if someone came along to show evidence that this antibody is not specific.