IP3R Immunofluorescence

Food Science and Human Nutrition
Colorado State University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Santa Cruz Biotechnology

Product Name:

IP3R-1 Antibody (E-8)

Catalog Number:

sc-271197

The interaction between the ER and mitochondria has been shown to be involved in the development and progression of obesity and obesity-related disorders such as non-alcoholic fatty liver disease. IP3R is a protein associated with the ER and has been shown to interact with proteins of the mitochondria. This experiment was performed to identify IP3R using immunofluorescence in liver cells.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

H4IIE rat heptoma cells

Primary Incubation

1:50 antibody in 1% BSA in PBST for 1 hr. 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 Alexa Fluor dye 555 in 1% BSA in PBST for 1 hr. at room temperature in the dark

Tertiary Incubation

N/A

Detection

Olympus BX63 with Cy3 filter

Results Summary

IP3R seems to appear in the nucleus. It still appears outside of the nucleus but in considerably lower quantities. Not sure if this antibody is interacting with the DAPI nuclear stain or if too much antibody was used. Will have to continue to optimize or get a different antibody from somewhere else because this is not ideal and this protein should not be present in the nucleus.

Additional Notes

None

Related Categories

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Summary

The Good

Antibody is easily viewable

The Bad

A lot of non-specific binding to nuclear area and/or interacts with DAPI.

The Bottom Line

Consider increased experiments for optimization or choose an antibody from a different company.

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