WB PROTOCOL
Immunoblotting, also known as Western blot (WB), is a comprehensive immunological detection technique. It uses SDS-PAGE to separate protein molecules in biological samples according to their molecular weights on a gel, and then transfers the proteins to a solid phase membrane by electrotransfer, using the proteins on the solid phase membrane as antigens to react with the corresponding antibodies, and then with an enzyme-labeled secondary antibody, and then detects the proteins expressed by specific target genes separated by electrophoresis, such as by substrate chromatography or fluorescence imaging. The protein expressed on the membrane is used as an antigen and reacts with the corresponding antibody.
I. Electrophoresis:
1. Configure the SDS-PAGE gel according to the molecular weight (MW) of the target protein. (Prepared according to the recommendations and gel formulations presented in this article)
Tip a : Tris-tricine gel separates low molecular weight proteins (<10kDa) better than Tris-glycine gel.
Tip b : Gradient gels provide sharper bands and a larger protein detection range on one gel, e.g. 10-500kDa.
2. Prepare samples in EP tubes. Add 4X SDS sample buffer so that each sample contains 30-50 μg total protein (protein amount determined by Bradford or BCA protein assay).
3. Samples were vortexed and then heated to 95-100°C for 5 minutes.
4. Clean the residual gel in the well, place it in the electrophoresis tank and add diluted SDS-PAGE electrophoresis solution (1X).
II. Protein transfer (wet transfer example).
1. PVDF membranes are recommended (PSQ membranes with 0.22 μm pore size only if the molecular weight of the target protein is <20 kDa). Soak the membrane in methanol for 30 seconds, rinse twice with ultrapure water, and then soak in Western Blot Wet Transmembrane Solution. Also soak the filter paper and sponge in the Membrane Transfer Solution.
2. Remove the gel from the electrophoresis tank and soak in Membrane Transfer Solution.
3. Arrange them tightly in a sponge/filter paper/film/glue/filter paper/sponge fashion, making sure there are no air bubbles between the layers.
4. The constant current method (typically 200mA for 90min) was used for electrical rotation.
5. After transfer, the membrane was removed, rinsed twice with ultrapure water, and immersed in Western Blot Blocking Solution for two hours at room temperature or overnight at 4°C.
Tip c: If the target MW is larger than 150 kDa, it is recommended to wet transfer overnight at 4°C and it is best to add 0.005% SDS to the transfer buffer for better transfer of large proteins.
Tip d: If you need to observe protein transfer, stain the membrane with Lichun Red and wash with TBST after observation.
III. Immunoblotting:
1. The membrane was removed from the containment solution and washed three times with 1x TBST for 5 minutes each time.
2. Dilute the antibody with Antibody Diluent to the recommended dilution and incubate for 1 hour at 37°C or overnight at 4°C.
3. The membrane was washed three times with 1xTBST for 10 minutes each time.
4. Dilute the secondary antibody to the recommended dilution and incubate the membrane for 1 hour at 37°C.
5. The membrane was washed three times with 1xTBST for 10 minutes each time.
6. Rinse the membrane twice with ultrapure water.
Tip e: Keep the membrane wet at all times during the experiment.
IV. Exposure:
1. Mix ECL Substrate Liquid A and Liquid B 1:1 in the ECL Chemiluminescence Detection Kit and use.
2. After the ECL substrate and membrane had fully reacted, the membrane was wrapped and secured to the dark clip with plastic wrap.
3. Expose the membrane to dark photographic film or read with a chemiluminescent imager.
Tip f: Determine the optimal exposure time based on multiple exposure times.
4. Make notes on the exposed film with information such as: exposure time, lane, and sample name.
V. Related Reagent Formulations
4X SDS-PAGE Protein Sample Buffer: 1 M Tris-HCl (pH 7.0) Masterbatch 150 ml, glycerol 250 ml, SDS 120 g, Bromophenol Blue 0.4 g. Fill to 800 ml with ultrapure water and store at -20°C.
Add 2M DTT solution (orβ-mercaptoethanol) at a volume ratio of 4:1 before use.
1x TBST (washing solution)
Tris 2.42 g, NaCl 8.76 g, KCl 0.373 g, Tween-20 500 ul. Adjust pH to 7.4 with concentrated hydrochloric acid; make up to 1000 ml with ultrapure water).
Wet Transfer Fluid
Tris 2.9 g, Glycine (Gly) 14.5 g, ethanol 200 ml, Fill to 1000 ml with ultrapure water.
Semi-dry transfer buffer (for Tris-Tricine gel transfer)
Tris 36.3 g, Acetic acid 6 ml, methanol 200 ml, Fill to 1000 ml with ultrapure water.
VI. SDS-PAGE Gel Formulation
1. Traditional Adhesive Formulations
For conventional proteins larger than 10 kDa, use the following SDS-PAGE gel formulation for preparation, selecting the appropriate gel concentration based on the molecular weight size. The formulations are as follows:
Separator Gel(10 ml) | ||||
Molecular weight of the target protein (kDa) | 80-200 | 25-100 | 20-40 | 10-25 |
Gel Concentration | 8% | 10% | 12% | 15% |
ddH₂O | 3 | 2.5 | 2 | 1.25 |
40% Acrylamide Masterbatch | 2 | 2.5 | 3 | 3.75 |
2x Separation Gel Buffer Masterbatch | 5.0 | 5.0 | 5.0 | 5.0 |
10% Ammonium persulfate mother liquor | 0.1 | 0.1 | 0.1 | 0.1 |
TEMED | 0.01 | 0.01 | 0.01 | 0.01 |
Concentrated gel | 4 ml | 6ml | 8ml |
Molecular weight of the target protein (kDa) | - | - | - |
Gel Concentration | 4% | 4% | 4% |
ddH₂O | 1.6 | 2.4 | 3.2 |
40% Acrylamide masterbatch | 0.4 | 0.6 | 0.8 |
2x Gum buffer concentrate mother liquor | 2.0 | 3.0 | 4.0 |
10% Ammonium persulfate mother liquor | 0.04 | 0.06 | 0.08 |
TEMED | 0.004 | 0.006 | 0.008 |
2x Separation Gel Buffer(1000ml) |
|
Tris | 90.8 g |
SDS | 2.0 g |
After sufficient dissolution, the pH was adjusted to 8.8 with concentrated hydrochloric acid and the volume made up to 1000 ml with ultrapure water. | |
2x Concentrated Gel Buffer (1000 ml) |
|
Tris | 30.3 g |
SDS | 2.0 g |
After sufficient dissolution, the pH was adjusted to 6.8 with concentrated hydrochloric acid and the volume made up to 1000 ml with ultrapure water. | |
1x SDS-PAGE Electrophoresis Buffer ( 1000 ml) |
|
Tris | 3.03 g |
glycine (Gly) | 18.75 g |
SDS | 1 g |
After sufficient dissolution, the volume was made up to 1000 ml with ultrapure water. | |
2. Tris-Tricine Gel Formulation
For molecular weights below 10 KDa, the Tris-Tricine gel system is recommended. The formulation is as follows.
Concentrated gel 4%, Gap Adhesive 10%, Separator Gel 15%.
Reagents | Concentrated Gel | Gap Adhesive | Separation Gel |
Gel Concentration | 4% | 10% | 15% |
Volume | 2 ml | 1.5 ml | 6 ml |
37% Glycerin Masterbatch | - | - | 1.63 ml |
ddH₂O | 1.51 ml | 0.595 ml | - |
40% Acrylamide masterbatch | 0.2 ml | 0.375 ml | 2.25 ml |
3M Tris HCl (pH 8.5)masterbatch | - | 0.5 ml | 2.0 ml |
1M Tris HCl (pH 6.8)masterbatch | 0.25 ml | - | - |
10% SDS masterbatch | 0.02 ml | 0.15 ml | 0.06 ml |
10% APS masterbatch | 0.02 ml | 0.015 ml | 0.06 ml |
TEMED | 0.002 ml | 0.0015 ml | 0.003 ml |
1x Tris-Tricine Cathodic Electrophoresis Buffer ( 1000ml ) |
|
Tris | 12.1 g |
Tricine | 17.9 g |
SDS | 1 g |
After sufficient dissolution, the volume was made up to 1000 ml with ultrapure water. | |
1x Tris-Tricine Anodic Electrophoresis Buffer ( 1000ml ) |
|
Tris | 24.23g |
After complete dissolution, the pH was adjusted to 8.9 with concentrated hydrochloric acid and the volume made up to 1000 ml with ultrapure water. | |