western blot protocols, troubleshooting, scientific resources and research atricles for the research community and science students
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| Web | www.westernblotting.org |
SDS-PAGE is a commonly used molecular biology protocol that allows for the separation of molecules (i.e. the proteins for Western Blotting). Proteins are separated based on weight and electrical properties as they migrate through a polyacrylamide gel matrix. Acrylamide gel preparation is a process that involves the crosslinking of acrylamide monomers with the use of catalysts. Once the gel has set, protein samples can be loaded into the gel and separated along an electric field.
1. PubMed:
comprehensive database - 15 million citations
2. NIH:the National Institutes of Health
3. UniProt: The world's most comprehensive catalogue of information on proteins
4. Western
Blot Procedure Western blot protocol overview.
b. With a 10 well mini gel, a volume (including sample, buffer, beta mercaptoethanol) of no more than 50 µl is recommended for each lane.
c. Add 10-15 µl of Laemmli buffer to a microtube.
d. Add beta mercaptoethanol. (5% of total volume).
e. Add a protein sample volume that will contain 100 – 150 µg of protein to the tube with the other reagents.
f. Heat the microtubes at 70 degrees Celsius for 2 minutes, then place on ice
b. Position the acrylamide gels in the gel holder assembly and immerse into the tank.
c. Fill the inner compartment (between the two gels) with SDS-PAGE Buffer.
d. Carefully load the samples in the wells (using a fine-tipped pipette).
e. Place the lid on the tank and plug it into the power source. (Note: this procedure uses electric current to separate proteins – use caution when working with tank electrophoresis to avoid injury).
f. Run the apparatus at 100V until the samples have passed the stacking gel.
g. Turn the voltage up to 160V and allow the samples time to separate; use a pre-stained molecular weight marker to determine the end-point of the electrophoresis.