VDAC For Proximity Ligation Assay

January 30, 2017

Food Science and Human Nutrition
Colorado State University
Graduate Student

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Quality of Results

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Company:

Abcam

Product Name:

Anti-VDAC1 / Porin antibody

Catalog Number:

ab14734

The mitochondrial associated membrane (MAM) is now an established point of communication between the ER and mitochondria. VDAC is typically found in the mitochondria but has been shown to interact with IP3R, a protein typically found in the ER. Both of these proteins have been isolated in MAM fractions.The goal of this experiment was to find a working VDAC antibody for fluorescent microscopy to ultimately be able to use in a proximity ligation assay with IP3R to observe MAM dynamics.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Fixed and permeabilized H4IIE rat hepatoma cells

Primary Incubation

1:200 VDAC 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-Mouse Alexa Fluor 555 in 1% BSA in PBST for 1 hour at room temperature

Tertiary Incubation

N/A

Detection

Olympus BX63 with motorized stage

Results Summary

Using Abcam's immunofluorescence protocol, this antibody works really well. Exposure times were in the millisecond range and there was very minimal background as VDAC was clearly bright red. Could probably get away with using a higher dilution.

DOI or PMID #

N/A

Additional Notes

N/A

Related Categories

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Summary

The Good

Antibody worked on the first go around. Strong signal. No background.

The Bad

Expensive!

The Bottom Line

Although Abcam is relatively expensive, their antibodys work. I have found very few from Abcam that don't work and I have returned them with a full refund or exchanged them with little hassle. Don't bother trying another antibody because although pricey, this one works well.

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