Phosphorylated IRE1alpha

August 25, 2016

Food Science and Human Nutrition
Colorado State University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Abcam

Product Name:

Anti-IRE1 (phospho S724)

Catalog Number:

ab48187

IRE1alpha is a key protein that is phosphorylated and activated during ER stress, initiating the unfolded protein response. Determining the activation state of IRE1alpha due to a variety of stress conditions is helpful in assessing the severity of ER stress.

Experimental Design and Results Summary

Applications

Western Blot

Sample

H4IIE rat hepatoma liver cells

Primary Incubation

1:1000 in 1:3 odyssey block:1X TBST (0.1% Tween) shaking, overnight at 4 degrees C

Blocking Agent

1:3 odyssey block:1X TBS at room temperature for 1 hr.

Secondary Incubation

1:15000 Goat anti-rabbit IRDye in 1:3 odyssey block:1X TBST (0.1% Tween) and 0.2% SDS for one hour at room temperature in the dark

Tertiary Incubation

N/A

Detection

Licor Odyssey

Results Summary

Similar to total IRE1alpha from Abcam, phospho IRE1alpha shows up at around 125kDa, but the rest of the blot is pretty messy. Lots of background and prominent bands around 70kDa and 25kDa that skews the blot so that you can't initially see phospho-IRE1alpha until the brightness and contrast have been adjusted.

Additional Notes

None

Related Categories

Image Gallery

Summary

The Good

Easily detectable.

The Bad

Lots of background

The Bottom Line

Although the blots aren't the greatest and there is a lot of background, this antibody does show up and is easily quantifiable. Can't beat Abcam's guarantee either. If an antibody doesn't work, they will refund your money or send you a new one.

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