LC3B Antibody for I/II Ratio

Immunology
University of Pittsburgh
Lab Technician

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
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Company:

Cell Signaling Technologies

Product Name:

LC3B XP® Rabbit mAb

Catalog Number:

3868

Clone Number:

(D11)

We wanted to be able to quantify the ratio of LC3B-I and LC3B-II. The sizes are 16 and 14 kDa respectively and are quite hard to resolve these two bands. With some modifications to our normal blotting protocol, we were able to achieve these results.

Experimental Design and Results Summary

Applications

Western Blot

Sample

BMDMs

Primary Incubation

1:1000 overnight at 4 degrees C

Blocking Agent

5% BSA

Secondary Incubation

1:3000 anti-rabbit HRP linked for 1 hour at room temp

Tertiary Incubation

None

Detection

SuperSignal™ West Femto Maximum Sensitivity Substrate

Results Summary

We were able to resolve the 2 bands after optimizing our protocol.

DOI or PMID #

N/A

Additional Notes

We used 8-16% gels and ran until 1the 10 kDa marker was nearly off the gel (the loading dye had run off). We used a rapid transfer optimized for low MW proteins.

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Summary

The Good

Good detection, clean blot

The Bad

Can be hard to optimize

The Bottom Line

With some modifications to our normal blotting protocol, we were able to identify and resolve the two types of LC3B in order to quantify the ratio.

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