Western Blot
Background
Western Blot is a technique that uses antibodies for the specific recognition of a chosen antigen in a solution containing numerous proteins and other components, relying on the separation of the specific antigen from other substances based on molecular weight and the specificity of antibody-antigen recognition. Western Blot relies on the standard methods of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In general, SDS-PAGE is conducted in flat minigels, using either reducing or non-reducing buffers based on the presence or absence of disulfide bond breaking reagents such as beta-mercaptoethanol or dithiothreitol.
Direct Western Blot is applicable for primary antibodies already conjugated with a detection system. Direct Western Blot has advantages in that the method is rapid because only one antibody is used. Any cross-reactivity of the secondary antibody is also eliminated. The disadvantages of this method are 1) the affinity of the primary antibody may be reduced by conjugation and 2) there is little signal amplification. Indirect Western Blot is the more frequently used application. The advantages of this method are: 1) the reactivity of the primary antibody is preserved, 2) a wide variety of secondary antigen conjugations are available, and 3) sensitivity is increased by multiple binding of the secondary.
Indirect Western Blot requires a secondary antibody bearing the conjugate of choice for detection. The disadvantages of indirect Western Blot include the fact that cross-reactivity between the secondary antibody and other proteins can lead to high background signals, and the extra step takes additional time.
Direct Western Blot
Materials
Transfer Buffer: 25mM Tris base, 0.2M glycine, 20% methanol, pH 8.5.
SDS Sample Buffer (1X): 62.5mM Tris-HCl, pH 6.8 at RT, 2% SDS, 10% glycerol, 50mM DTT, 0.01% bromophenol blue or phenol red.
10X TBS: To prepare 1 liter of 10X TBS: 24.2g Tris base, 80g sodium chloride. Adjust pH to 7.6 with HCl. Use at 1X.
Blocking Buffer: 1X TBS, 0.1% Tween-20, 5% nonfat dry milk. For 150ml, add 15ml 10X TBS to 135ml water, mix. Add 7.5g nonfat dry milk and mix well. While stirring, add 0.15ml Tween-20 (100%).
Wash Buffer TBS/T: 1X TBS, 0.1% Tween-20
Sample Preparation and Separation
1.Boil ~20ug sample in SDS sample buffer for 5 minutes and resolve on SDS-PAGE (12-15%).
2.Transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.
Blocking Step
3.Block the membranes for 1 hour at RT with 5% nonfat dry milk in Tris-buffered saline-Tween (TBS-T) for 20 minutes at RT with constant agitation.
4.Wash 3 times in TBS-T.
Primary Antibody Incubation
5.Incubate the membrane with the primary antibody (diluted in blocking buffer) for 2 hours at RT or overnight at 4°C.
6.Wash 3 times in TBS-T.
Color Development
7.Detect using enhanced chemiluminescence detection method.
Indirect Western Blot
Materials
Transfer Buffer: 25mM Tris base, 0.2M glycine, 20% methanol, pH 8.5.
SDS Sample Buffer: 62.5mM Tris-HCl, pH 6.8 at RT, 2% SDS, 10% glycerol, 50mM DTT, 0.01% bromophenol blue or phenol red.
Blocking Buffer: 1X TBS, 0.1% Tween-20, 5% nonfat dry milk.
Wash Buffer TBS/T: 1X TBS, 0.1% Tween-20
For secondary antibody, select a product directed at the isotype of the primary antibody (anti-mouse IgG1, anti-rabbit IgM, anti-chicken IgY, etc.); conjugate as desired.
Sample Preparation and Separation
1.Boil ~20ug sample in SDS sample buffer for 5 minutes and resolve on SDS-PAGE (12-15%).
2.Transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.
Blocking Step
3.Block the membranes for 1 hour at RT with 5% nonfat dry milk in Tris-buffered saline-Tween (TBS-T) for 1 hour at RT with constant agitation.
4.Wash 3 times in TBS-T.
Primary Antibody Incubation
5.Incubate membrane with primary antibody (diluted in blocking buffer) for 2 hours at RT or overnight at 4°C.
6.Wash membranes 3 times in TBS-T.
Secondary Antibody Incubation
7.Dilute secondary antibody in TBS-T with 1% nonfat dry milk and incubate for 1 hour at RT.
8.Wash 3 times for 5 minutes each in TBS-T.
Color Development
9.Detect using enhanced chemiluminescence detection method.