Decent Cleaved Caspase-3 Antibody From Cell Signaling for Western Blot

October 09, 2023

IKP/RBCT
Robert Bosch Gesellschaft für medizinische Forschung mbH
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Cell Signaling

Product Name:

Cleaved Caspase-3 (Asp175) Antibody

Catalog Number:

9661

Clone Number:

Polyclonal rabbit

I used this antibody several times for the detection of apoptosis induction in whole-cell lysates of prostate carcinoma cells. The detection did not work right away and needed to be optimized. However, the results were satisfying.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Human cancer cell lines (whole cell lysates) e.g. DU145

Primary Incubation

1:1000 at 4°C overnight

Blocking Agent

5 % BSA in TBST

Secondary Incubation

1:2000, 60 min at RT

Tertiary Incubation

None

Detection

HRP, Chemiluminescence (West Dura Extended Duration Substrate) in Elysia-raytest detector (3 min exposure)

Results Summary

ECL detection yielded visible bands in the molecular range of about 17-19 kDa (cleaved Caspase-3 fragment). No background or unspecific bands are detectable in the range analysed.

DOI or PMID #

-

Additional Notes

I recommend to start at a higher concentration of 1:500 initially. The antibody only results in decent signals for a high amount of protein and/or cell death in the sample. Make sure to include a positive control.

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Summary

The Good

No background/unspecific bands.

The Bad

Optimization for different samples is needed.

The Bottom Line

This antibody is suitable for detection of cleaved caspase-3 via Western Blot. Other applications were not tested. As a more regular used alternative I also use anti-PARP antibody (CST9542) for apoptosis detection which usually gives stronger signals than this antibody.

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