Hippocampal Cell Lysate WB for RNA Pol II

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Anatomy and Neurobiology
University of California Irvine
Post-doctoral researcher

Company:

Millipore

Product Name:

Mouse anti-RNA Polymerase II

Catalog Number:

05-952

Stress in vivo, and CRH application in vitro, cause dendritic spine loss in hippocampal pyramidal neurons. This loss of spines, and subsequent synapses, is likely the underlying mechanism through which severe stress disrupts learning and memory. CRH, in combination with neuronal activity and NMDA receptor activation, recruits the actin destabilizing protein, calpain, to break down the actin cytoskeleton resulting in spine loss. Identifying the downstream signaling from CRH receptors could reveal potential therapeutic targets for diseases such as post-traumatic stress disorder (PTSD), anxiety, and depression.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Whole cell hippocampal cell lysate

Primary Incubation

1:2000

Blocking Agent

10% milk in PBS-Tween

Secondary Incubation

1:10,000

Tertiary Incubation

None

Detection

Thermo Fisher ECL Kit CAT#PI80196

Results Summary

I performed a pull down for membrane bound CRH receptor type 1 and ionotropic glutamate receptors and needed a nuclear protein as a negative control.I visualized using an ECL kit and developed on Amersham Hyperfilm (GE Healthcare CAT#28906837) for 12 mins, 8 mins, 6 mins, and 4 mins. The 10 minute exposure has unspecific bands but the 8 minute exposure produced 2 clear bands greater than 180kDa. The expected MW of RNA Pol II is 220kDa.

Additional Notes

None

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Summary

The Good

Produced a clean band at expected molecular weight

The Bad

Unspecific bands visible at higher exposure times

The Bottom Line

Good antibody for RNA Pol II WB in hippocampal cell lysate. Have also tried in other brain areas such as cortex and forebrain and also produces specific and reliable band.

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