Posted on June 1st, 2016 by UNM CC
(Includes sample preparation, Bradford Assay, SDS-PAGE, semi-dry transfer, antibody staining and ECL development.)
The chief goal is to determine the relative protein abundance across samples to ensure equivalent loading of samples on the gel. A second goal is to ensure that the absolute quantity of protein loaded is in a range that is sufficient for visualization but does not exceed the capacity of the gel.
If an abundant complex mixture is being assayed (e.g. cell or tissue extracts) then an A280 measurement may suffice. However, A280 measurements are dependent on the amino acid composition of the sample, chiefly tryptophan and tyrosine, so A280 readings may not be appropriate for adjusting the concentrations of two different purified proteins or two different tissue types. A280 readings are sensitive to the presence of contaminating nucleic acids (DNA and RNA).
The Bradford assay is relatively easy, sensitive, and less dependent on amino acid composition. It has a limited linear range, so it should be repeated on samples that give high readings and are subsequently diluted. It is recommended to measure all samples, that will be run on a single gel, together in a single Bradford assay, since there may be day to day variability in the results.
SDS PAGE (Volumes used for 1 mm BioRad Mini-Protean gel system)
|Stack Gel (4 mL)||Separating gel (10 mL)|
|MW Range (kDal):||-||60 - 200||16 - 70||14 - 60||12 - 45|
|30% Acrylamide mix
|0.67 mL||1.7 mL||3.3 mL||4 mL||5 mL|
|1.5M Tris pH8.8||-||2.5 mL||2.5 mL||2.5 mL||2.5 mL|
|1M Tris pH6.8||0.5 mL||-||-||-||-|
|H2O||2.4 mL||5.7 mL||4.1 mL||3.4 mL||2.4 mL|
|10% SDS||40 µL||100 µL||100 µL||100 µL||100 µL|
|10% ammonium persulfate||30 µL||50 µL||50 µL||50 µL||50 µL|
|TEMED||3 µL||5 µL||5 µL||5 µL||5 µL|
(Tris buffers must be made from Tris-base, and are pH'd with conc. HCl. Store acrylamide, 10% APS, and TEMED at 4ºC.)
Note: Following electrotransfer a small amount of protein (~10% of the total) will remain in the gel, assuming a 1mm gel was blotted for the time listed above. If a thinner gel is used then only a small amount of high MW material may remain or else the electrotransfer time may be proportionally reduced. Having a small amount of residual protein in the gel is convenient because it can be post-stained with Coumassie blue in order to document consistent protein loading across samples. Alternatively, membranes may be stained with Ponceau S solution (Sigma) for the same purpose.
Western blots are usually not quantified and thus are limited to qualitative interpretations, e.g. "The amount of protein X is higher in sample 1 than in sample 2", or "The levels of protein Y is unchanged across the samples". Still, some investigators choose to immunostain blots a 2nd time with an antibody against a protein, such as actin or tubulin, which are expressed at stable levels, as a "protein loading control". This gives visual reassuance that the changes seen for the protein of interest was not due to technical problems with protein loading. Regardless, of whether such a control is used, researchers should establish that both the experimental protein (and the loading control) are expressed within the "linear range" of the assay. For example, ECL may exhibit a threshold effect wherein the reduction of the target protein below a certain level is associated with a disappearance of a band rather than a reduction of its intensity. The detection system may also become "saturated" for proteins with high expression levels, such that significant differences in levels will appear to be the same. This is often the case for actin loading controls if a high concentration of antibody is used.
Western blots can be quantified with a reasonable level of accuracy if one is careful about the technical setup. A traditional way to do this is to scan films and perform densitometry. Fluorescent antibodies can also be used, in conjunction with an Odyssey imager, and exhibit a greater dynamic range and accuracy than densitometry. In either case, a standard curve comprised of two-fold serial dilutions of a positive control should be run in parallel to verify the linearity of the assay and to quantify the results. Minor differences in protein loading may be normalized by the differences seen in the actin or tubulin internal control, assuming that this too exhibits a linear relationship in a standard curve.
|RIPA cell lysis buffer||TG cell lysis buffer|
|10 mM NaPO4, pH7.2||20 mM HEPES, pH7.2|
|0.3 M NaCl||1% Triton-X|
|0.1% SDS||10% glycerol|
|1% DOC (deoxycholate)|
|2 mM EDTA|
Leupeptin 10 mg/mL (1000x) Store at -20°C.
Aprotinin 10 mg/mL (1000x) Store at -20°C.
PMSF: Phenylmethylsulfonyl flouride, 200mM in ethanol (100x) . Store at 4°C.
100x Phosphatase inhibitors (for kinase assays)
100 mM NaF
50 mM NaVanadate
800 mM ß-glycerol phosphate
|5x Laemmli sample buffer (15 mL)||1x Concentrations|
|1.5 gm SDS||2% (w/v)|
|3.75 mL 1M Tris, pH 6.8||50 mM|
|0.015 gm bromphenol blue||0.2 mg/mL|
|1.16 gm DTT||0.1 M DTT|
|q.s. to 7.5 mL with H2O||-|
|7.5 mL Glycerol||10% (v/v)|
|10x SDS Running Buffer (8L)||1x Concentration|
|1440 g Glycine (75 g/mole)||250 mM|
|242 g Tris-Base (121 g/mole)||25 mM|
|80 g SDS (electrophoresis grade)||0.1% (w/v)|
|q.s. to 8 L with H2O|
Western Transfer Solutions
Solution A: 25 mM Tris Base, 20% v/v isopropanol, 40 mM e-aminocaproic acid.
Solution B: 25 mM Tris Base, 20% v/v isopropanol.
Solution C: 250 mM Tris Base, 20% v/v isopropanol.
Coomassie Stain and Destain
Coomassie Stain: 0.25% w/v brilliant blue (Sigma B-0770), 50% v/v methanol, 7.5% v/v glacial acetic acid. Filter through Whatman #1.
Destain: 10% (v/v) methanol, 10% (v/v) glacial acetic acid.
0.5% TNT: 0.5% Tween-20, 0.15 M NaCl, 25 mM Tris pH 7.4.
Non-fat dried milk.
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