I would like to test for ubiquitination in my samples, but don't want to end up with a smeared blot. Can anyone recommend a good antibody to use? Or does any company make anti-ubiquitin MKP-1 specific antibodies?? I looked, but could not find anything
Thank you!! :)
Hi Sarah,
If I understand you right, you would like to test if your protein of interest is ubiquitylated or not? There are various ways of doing this. Modification of a protein with ubiquitin is usually very labile, and a small percentage of your protein is normally detected as modified. Since you would like to probe with an anti-ubiquitin antibody, I take it that you probably have IPed or enriched for your protein in some way? If this is the case, it is very important to include inhibitors of de-ubiquitylating enzymes in your lysis buffer to prevent your protein from rapid demodification (chloroacetamide or NEM for example).
You also mention that you do not want to end up with a smear on your MKP-1 blot. I am unsure why this would be a problem, as this would be strong evidence for ubiquitylation of your protein (assuming a prior step of MKP-1 IP or similar).
As a first step towards showing that your protein is actually ubiquitylated, I would suggest to do a pulldown using His6-ubiquitin. This can be overexpressed in the cell model of your choice, followed by lysis under denaturing conditions (see e.g. Andersen, J., Matic, I. & Vertegaal, A. Identification of SUMO target proteins by quantitative proteomics. Methods Mol Biol 497, 19–31 (2009)) and a Ni:NTA purification. This method will enrich for all cellular proteins modified by ubiquitin, and associated proteins will be lost due to the denaturing nature of the buffers. You may then probe for your protein of interest, which should mainly appear as high molecular weight forms, either as a smear or as a ladder of discrete bands, where each band represents an additional ubiquitin moiety.
To follow up on this, there are methods to isolate ubiquitylated proteins without overexpression of tagged ubiquitin (Hjerpe et al. EMBO Rep 2009) which can be useful to solidify the results.
Finally, there are various good ubiquitin antibodies - the following one from DAKO usually works well:
http://www.dako.com/uk/ar38/p106260/prod_products.htm?setCountry=true&purl=ar38/p106260/prod_products.htm
Another option is Mab1510 from Millipore: http://www.emdmillipore.com/INTL/en/product/,MM_NF-MAB1510
Good luck!
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