Customer Experience using HSP60 Antibody

Mon, 04/30/2012 - 10:50

I began using the HSP60 antibody (NB110-57063) in June of 2010 and it worked well. I do not like to buy antibodies that have not been tested in the species for which I will use them, so I picked this antibody because it had already been tested in rat tissue. I split the antibody into 20ul aliquots and stored it at -20C. I first ran a Western blot with 15ug of a RIPA whole cell lystate from WKPT cells a rat kidney immortalized cell line derived from the S1 proximal tubule segment.

Western Blot: Hsp60 Antibody (EP1006Y) [NB110-57063]

I used a 10% SDS-PAGE gel and transferred to PVDF membrane for 1 hour in transfer buffer with 10% methanol. I then blocked with odyssey blocking buffer for 2 hours at RT.  I probed with the primary antibody with a 1:1000 dilution over night at 4C, and have even gotten good signal with as little as 1:5000 dilution of the antibody. I incubated 1 hour with Licor goat anti rabbit 680nm secondary then imaged on an Odyssey scanner. I saw a band at about 60kDa, the blot was clear of any other containment bands. I used this antibody several times with similar result.  I have only tried it with WKPT samples so far, but it worked great and I would recommend this antibody to others. I recently reused it and found I needed to use a more concentrated dilution (ie. 1:1000), however this is likely because it has been stored in -20 C for a year and a half.

This guest blog was submitted by Novus customer, Dana Freund of Colorado State University.

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