Please consult our separate protocols for sub-cellular fractionation.
This site uses Akismet to reduce spam. Take careful note of these terms before you buy a Western blot antibody check the antibody datasheet for words like mild detergent or without detergent, or run a quick full text search with your antibody in our full text search engine. These tend to aggregate when boiled and the aggregates may not enter the gel efficiently.
NP40 buffer — good for antibodies that react to non-denatured proteins 150 mM NaCl 1. The minimum recommended concentration is 0. This is a popular buffer for studying proteins that are cytoplasmic or membrane-bound, or for whole cell extracts. Repeat this cycle until the solution remains at pH 9. Thanks for this article. Therefore, migration is determined by molecular weight, rather than by the intrinsic charge of the polypeptide.
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.
You can determine the purity of your extracts by western blotting for markers that are known to specifically reside in each of the fractions. Is the NP40 in the cytoplasmic buffer sufficient to solubilize them, or will you find them in the nucleus fraction? Storage Phosphor Screen. SDS grade is important for high-quality protein separation: As with all cell extraction protocols, you need to perform extractions on ice and with protease and phosphatase inhibitors.
Bring up to the initial volume with water.
Thanks for your help! Most antibodies will recognize reduced and denatured proteins and meaning your protein should be kept under reducing and denaturing conditions Duh. Get resources and offers direct to your inbox Sign up. NP-40 is a good general buffer for studying proteins that are easily released from cells, cytoplasm, or other proteins. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice.
Cytoplasm-specific markers include heat shock proteins which are normally found in mitochondria and should not be present in nuclear fractions and vimentin a cytoskeleton component. Primary antibodies for WB. Chemical Nomenclature Woes.
Protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. As a result, many scientists are blindly following kit instructions without knowing why certain steps are performed. Minimize volume change due to evaporation by covering loosely. During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution.