The Western Blot – a tried and true experimental protocol where protein structures are separated via molecular weight/charge and transferred to a membrane before visualization by a chemiluminescent solution (say that three times fast!). Seems simple, right? While the step-by-step process of a western blot has for the most part remained the same over the years, variations in solutions, procedures and reagents may increase the efficacy of your results.
Western blotting is one of the most commonly used antibody assay techniques in cell and molecular biology research since its development over three decades ago, and is considered the gold standard for protein detection and quantification.
The Western blot is one of the most commonly used antibody assay techniques in cell and molecular biology research since its development over three decades ago, and is considered the gold standard for protein detection and quantification. The traditional Western blot can be a labor-intensive and time-consuming process, leading many researchers to seek an alternative method that is more efficient, reproducible and quantitative.
A growing body of data and studies using actin antibodies supports a view of the actin cytoskeleton of smooth muscle cells as a dynamic structure that plays an integral role in regulating the development of mechanical tension and the material properties of smooth muscle tissues.
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.
I first tried the PBP antibody (NB110-93495) in June of 2010 and it worked well. I picked this antibody because it had been tested in rat tissue, so I was confident it would work for my rat samples. I stored the PBP antibody in 20ul aliquots after it arrived then stored it at -20C and used it over a 3 month period. I 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 blotting is an essential technique to probe protein expression in complex cell or tissue lysates. To accurately determine protein expression and interpret Western blot results, it is important to use loading controls. A loading control antibody helps determine if samples have been loaded equally across all wells and confirms effective protein transfer during the western blot protocol.
The loading controls on our antibody database are widely used in gel electrophoresis and Western blotting studies. Products like the GAPDH antibody detect "housekeeping" proteins which are abundantly distributed in cells. This makes them useful for checking the even loading of gel samples, and the even transfer of proteins at the blotting stage. They also serve a purpose in quality control, by verifying reagents are working correctly, and in the standardization of experimental results.
The vast majority of antibodies in our antibody catalog are suitable for Western blotting studies. Devised almost 30 years ago by W. Neal Burnette, it has become a standard assay wherever antibodies are used to detect proteins.
Western blotting combines gel electrophoresis with use of a membrane to separate and identify target proteins using antibodies. Proteins are separated into bands using electrophoresis, and are then transferred to a membrane using filter-paper capillary action or an electroblotting technique. The effectiveness of the transfer can be checked by means of a stain – typically Ponceau S.