Western Blot for P65 (NF-kB) K310 Acetylation Stem Cells Whole Cell Lysate

March 19, 2020

Laboratory of t-RNA Transcription
Centre for Cellular and Molecular Biology
Research Associate

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Company:

Cell Signaling Technology

Product Name:

Acetyl-NF-κB p65 (Lys310)

Catalog Number:

3045S

Clone Number:

Polyclonal

Our aim is to examine the levels of P65 K310 acetylation in cancer stem cells treated with TNF-alpha. 1 million sphere cell lines were seeded in 6 well plate. 24-h post seeding cells were treated with TNF-a (20ng/ml) for one hour. Cells were lysed in RIPA buffer and loaded on 10% SDS PAGE. Transferred the proteins on to PVDF membrane and probed with anti-acetyl K310 antibody

Experimental Design and Results Summary

Applications

Western Blot

Sample

Cancer stem cell whole cell lysates

Primary Incubation

1:1000 in 5% BSA, overnight in cold room

Blocking Agent

5%Milk

Secondary Incubation

1:10000 in 5%milk

Tertiary Incubation

N/A

Detection

Chemi doc, femto reagent

Results Summary

We observed induction of P65 K310 acetylation at molecular weight correspondind to 65KD in the presence of TNF-alpha when compared with untreated cells.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Good antibody for western blot

The Bad

N/A

The Bottom Line

Overall a good antibody for western blot analysis. Few non-specific bands observed which might be due to low levels of acetylation in our cell lines, and also might be due to use of femto reagent.

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