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Yes, I used 8M Urea for bovine heart mitochondrial lysate as well.
Asked on Oct 18 2012
Thank you for your reply.
The lab let me know in the meantime that they think you have used too much bovine heart mito lysate. Usually, the loading amount for the bovine heart mito lysate should be at least one tenth of the cell lysates. The band shows up at the correct MW, the additional smear above could be due to overloading the well.
Also, the lab agrees that you will get better result if you use SDS-loading buffer instead of 8M urea for denaturing.
Our WB protocol contains the information about the SDS loading buffer:
"The standard loading buffer is called 2X Laemmli buffer, first described in Nature, 1970 Aug 15;227(5259):680-5. It can also be made at 4X and 6X strength to minimize dilution of the samples. The 2X is to be mixed in a 1:1 ratio with the sample.
Laemmli 2X buffer
0.004% bromophenol blue
0.125 M Tris HCl
Check the pH and bring it to pH 6.8.
When SDS is used with proteins, all of the proteins become negatively charged by their attachment to the SDS anions. SDS denatures proteins by “wrapping around” the polypeptide backbone. SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - i.e., the denatured polypeptides become “rods” of negative charge clouds with equal charge or charge densities per unit length.
In denaturing SDS-PAGE separations, therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. SDS grade is of utmost importance: a protein stained background along individual gel tracts with indistinct or slightly distinct protein bands are indicative of old or poor quality SDS. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size by using ß-mercaptoethanol or dithiothreitol (DTT).
Glycerol is added to the loading buffer to increase the density of the sample to be loaded and hence maintain the sample at the bottom of the well, restricting overflow and uneven gel loading."
The bovine lysate does not show a band around 85-90 kDa, thus indicating that the kit and antibodies work as expected. It might be that the way your samples were prepared differs from how the bovine lysate was prepared, thus resulting in the difference in MW of the Cox-1 bands.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Answered on Oct 18 2012