Calcium activity by measuring Phospho-CaMKII

September 24, 2013

Overall

Quality of Results

Ease-of-Optimization

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LMP
University of Toronto
Graduate Student

Company:

Cell Signalling Technologies

Product Name:

Phospho-CaMKII (Thr286) Antibody #3361

Catalog Number:

3361

Purpose of experiment was to look at intracellular calcium activity by measuring the activation of p- CaMKII. between two cell populations ( WT and KO MEFS). Lane 1, 4: Control Lane 2, 5: Calcium ionophore 1uM- 24hr Lane 3, 6: Calcium ionophore 2uM- 24hr Activation by a calcium ionophore was not clear as the baseline showed some signs of activity. Not really sure why this happened. Calcium signalling must be important in MEFS.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Murine Embryonic Fibroblast cells (10-15ug) per well

Primary Incubation

1/1000, RT for 1hr in TBST 5% skim milk

Blocking Agent

TBST 5% skim milk

Secondary Incubation

1/10000, RT for 1hr in TBST 5% skim milk

Tertiary Incubation

N/A

Detection

HRP- chemiluminescence

Results Summary

Antibody appears to either be no specific in MEFS as there is basal activation in the MEFS. Antibody was easy to optimized for WB but may need to try different stimuli to see a better activation of p-CamKII Will try in a different cell type that contains a lower background activation of p-CamKII.

Additional Notes

None

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Summary

The Good

Easy to use and works at a fairly dilute range

The Bad

Contains a number of bands near expected size..possibly cell type specific

The Bottom Line

Would try it again with a different cell line. Cell Signalling usually makes great Ab products.

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