Western blotting is probably the worlds most widely used biochemical method. It is based on three important and very well cited papers. The first two concern the technique of polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Laemmli showed that proteins could be reliably fractionated using this method, which he described in a figure legend in a Nature paper (Laemmli, 1970). This paper used the method to study specifically the proteins of bacteriophage T4. At about the same time Weber and Osborn showed that the method had general applicability and also showed that the relative mobility of proteins in SDS-PAGE gels correlated quite well with their molecular weights (Weber and Osborn 1969). Running protein gels became wildly popular, first in tubes, then as slabs and then as minigels. Then people began to appreciate that it was possible to transfer proteins out of SDS-PAGE gels onto nitrocellulose membranes and there stain them with antibodies. The most influential early paper in this area was that of Towbin et al. (Towbin et al 1979). Later studies used other kinds of membranes, notably the nylon like material PVDF, which allowed proteins transferred from SDS-PAGE gels to be subjected to direct peptide sequencing.
The reason that SDS-PAGE works is in overview, this; SDS is a powerful detergent, which has a very hydrophobic end (the dodecyl part) and highly charged part (the sulfate group). The dodecyl part interacts with hydrophobic amino acids in proteins. Since the 3D structure of most proteins depends on interactions between hydrophobic amino acids, the detergent destroys 3D structures, transforming them into linear molecules now coated with negative SDS groups. After boiling in SDS proteins therefore become elongated with negative charges arrayed down them, so they will move towards a positive electrode. It is not surprising that the largest ones are generally retarded the most by polyacrylamide gels, and the smallest ones the least. Since some proteins have few or no hydrophobic residues it is not surprising that such molecules don't run on SDS page in a fashion which accurately reflects their molecular weight. Modifications such as phosphorylation and especially glycosylation can also cause proteins to run more slowly than expected. Finally cross-linked proteins don't run as their molecular weight would predict, generally running slower particularly on higher percentage gels. However a particular protein runs on a particular
Below are formulae we have been using for years, the exact origins of which are lost in recent prehistory. They work about as well as any other methods that are around. There are generally two gels, the resolving gel and the stacking gel. The stacking gel is of very low acrylamide concentration and is used to form the wells into which the protein is loaded. The low acrylamide concentration also allows most proteins to be concentrated at the dye front.
The solutions are made up as below; All except the ammonium persulphate can be stored at room temperature for several months. Acrylamide solution may be light sensitive, so most people store in dark colored or aluminium foil covered bottle. The ammonium sulphate should be made up each week, and stored at 4 degrees C. Polymerization is quicker and more uniform if you degas the first three solutions for a few minutes in an Ehrlenmeyer flask prior to addition of the last three reagents.
Mix for about 20mls running gel solution, enough for two minigels
|Acrylamide/Bisacryclamide mix||10.8 mls||9 mls||8.1 mls||7.21 mls||6.76 mls||5.4 mls||4.5 mls|
|1M Tris/HCl pH=8.8||7.5 mls||7.5 mls||7.5 mls||7.5 mls||7.5 mls||7.5 mls||7.5 mls|
|Distilled water||1.4 mls||3.2 mls||4.1 mls||4.99 mls||5.44 mls||6.8 mls||7.7 mls|
|10% Ammonium persulfate||100ml||100ml||100ml||100ml||100ml||100ml||100ml|
Stacking Gel Solution, good for 2 minigels, ~4.8% acrylamide:
|1M Tris/HCl pH=6.8||1.25ml|
|10% Ammonium Persulfate||50ml|
Acrylamide/Bisacrylamide = 22.2g acrylamide, 0.6g bis-acrylamide (36.66:1 cross-linker ratio) to 100 ml water, filtered.
Reservoir/running buffer = 57.6g Glycine, 12g Tris, 4g SDS, water to 4 litres
Stain solution = 2.5g Coomassie Brilliant Blue R-250, 450 mls Methanol, 100 mls Glacial Acetic Acid, water to 1 liter.
Destain solution = 300 mls methanol, 400 mls acetic acid, water to 4 liters.
Sample buffer 5X; make up 100 ml and store away 5-10 ml aliquots.
|1M Tris/HCl pH = 6.8||31.25 ml|
|Bromophenol Blue (2% in ethanol)||750 ml|
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