Immunodetection of p-IkappaB-a in Mammalian Cell Line

Physiology
University of Tennessee HSC
Postdoctoral Researcher

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Company:

Santa Cruz Biotechnology Inc

Product Name:

p-IκB-α Antibody

Catalog Number:

sc-8404

Mouse pancreatic endothelial cell line was tested for time course effect of specific growth factor on IκB-α phosphorylation. Cells which were grown in Medium 131 containing microvascular growth supplements (MVGS) were growth arrested, treated with growth factor for different time periods, proteins were extracted and equal amounts of proteins were resolved in 10%SDS-PAGE followed by immunoblotting with p-IκB-α specific antibody obtained from Santa Cruz Biotech.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Mouse pancreatic endothelial cell whole cell lysate

Primary Incubation

Dilution (1:500) in 5% BSA prepared in TBST, overnight incubation at 4 degree Celsius

Blocking Agent

5% skimmed milk prepared in TBST

Secondary Incubation

Dilution (1:4000) in 5% skimmed milk prepared in TBST, 2hr at RT

Tertiary Incubation

NA

Detection

ECL detection in X-Ray film

Results Summary

Although, anti- p-IκB-α from SantaCruz biotech has detected a specific band of p-IκB-α at nearly 35 kDa (indicated low range protein marker, Bio-Rad# 161-0304), some nonspecific bands were also observed. Intensity of p-IκB-α band was fairly weak but time dependent increase in phosphorylation was detected.

Additional Notes

Weak detection of p-IκB-α band

Related Categories

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Summary

The Good

Detected p-IκB-α

The Bad

Antibody also cross-reacts with other nonspecific epitopes

The Bottom Line

Average antibody for detection

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