Immunofluorescence Of IP3R For Proximity Ligation Assay

January 31, 2017

Food Science and Human Nutrition
Colorado State University
Graduate Student

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Company:

Abcam

Product Name:

Anti-IP3 receptor antibody

Catalog Number:

Ab5804

The mitochondrial associated membrane (MAM) is a well established point of communication between the ER and mitochondria. IP3R is typically found in the ER but has been identified in isolated MAM fractions. It has been shown to interact with VDAC, a protein typically associated with the mitochondria.The overall objective of this experiment was to test this IP3R antibody for future use in a proximity ligation assay to observe the interaction between it and VDAC as a measure of MAM dynamics.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Fixed and permeabilized H4IIE rat hepatoma cells

Primary Incubation

1:200 antibody in 1% BSA in PBST for 1 hour at room temperature

Blocking Agent

1% BSA, 22.52 mg/mL glycine in PBST (PBS+ 0.1% Tween 20) for 30 min at room temperature

Secondary Incubation

1:200 goat anti-rabbit Alexa Fluor 488 for 1 hour at room temperature

Tertiary Incubation

N/A

Detection

Olympus BX63 with motorized stage

Results Summary

This antibody works. There is minimal background but the signal is a little weak. Bringing the exposure time up to 1-2 seconds helps but creates little background. It will be worth going back and adjusting the dilution to try and get a better signal. Still, this antibody will work well in experiments.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Antibody worked first try with clear results and minimal background

The Bad

Expensive and signal could be better. Will try a less diluted antibody

The Bottom Line

Abcam is a great company. Most antibodies I have gotten from them work really well. Even if they don't Abcam has always refunded me or exchanged the antibody with little hassle. I have tried one other IP3R antibody from santa cruz that did not work nearly as well as this antibody. Therefore, this antibody is worth the extra money.

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