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Questions and Answers
What is the amount of sample to be added per lane during gel loading?
What is the amount of sample to be added per lane during gel loading?
The transfer buffer for blotting should be stored in a refrigerator.
The transfer buffer for blotting should be stored in a refrigerator.
False
The gel should be run at ___________ V through the resolving gel.
The gel should be run at ___________ V through the resolving gel.
100V
What is the composition of the transfer buffer?
What is the composition of the transfer buffer?
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The nitrocellulose membrane should be equilibrated in 2 x transfer buffer.
The nitrocellulose membrane should be equilibrated in 2 x transfer buffer.
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The transfer sandwich should be created in the following order: _____________, Black Sponge, 2 Filter papers, Nitrocellulose membrane, _____________, 2 Filter papers, Black Sponge, _____________.
The transfer sandwich should be created in the following order: _____________, Black Sponge, 2 Filter papers, Nitrocellulose membrane, _____________, 2 Filter papers, Black Sponge, _____________.
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How long should the transfer process last?
How long should the transfer process last?
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What is the final step mentioned in the protocol?
What is the final step mentioned in the protocol?
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Study Notes
Western Blot Protocol
- The dye is used to check if protein has been transferred to the membrane.
- Membrane washing and detection steps:
- Wash membrane with water and detect with BioRad Imager.
- Cut membrane at 70kDa.
- Wash in 1x TBS-T until red staining is gone.
- Blocking and antibody incubation steps:
- Block membrane with 5% milk in TBS-T for 1h.
- Incubate membrane with primary antibody (Pank4 1:1000 rabbit 82 kDa, GAPDH 1:500 mouse 36 kDa) in blocking solution overnight at 4°C.
- Wash membrane 3x for 10min with TBS-T.
- Incubate membrane with secondary antibody (Goat anti-rabbit 1:5000, Goat anti-mouse 1:5000) in blocking solution for 1h at room temperature.
- ECL solution preparation and detection:
- Mix both substrates 1:1 (2ml per membrane) and incubate for 1min.
- Detect with BioRad Imager.
Buffer Preparation
- Various buffers with their components, concentrations, and comments:
- Bis-Tris buffer: Bis-Tris (209.24 g/mol), 1.25 mol/l, in aqua dest.
- 20x TBS buffer pH 7.6: Tris base (121.14 g/mol), 0.5 mol/l, NaCl (58.44 g/mol), 1.5 mol/l, in aqua dest.
- 1x TBS-T buffer: TBS buffer 20x, 0.1% Tween-20, dilute with aqua dest.
- 1x PBS buffer pH 7.4: NaCl (58.44 g/mol), 0.14 mol/l, KCl (74.55 g/mol), 0.0027 mol/l, Na2HPO4*2H2O (141.96 g/mol), 0.01 mol/l, KH2PO4 (136.09 g/mol), 0.0018 mol/l, in aqua dest.
Gel Preparation
- Gel preparation steps:
- Clean glass plates with ethanol, assemble the rack, and check with water if everything is tight.
- Add about 5mL of resolving gel solution.
- Carefully pipette 500µL of Isopropanol on top of the solution to remove air bubbles.
- Wait 30min, remove Isopropanol, overlay with running gel, and carefully insert the comb without air bubbles.
- Wait until the gel is solidified (about 30min).
SDS-PAGE
- SDS-PAGE steps:
- Mix sample with lämmli sample buffer according to calculated amounts after protein assay.
- Denature samples for 5min at 95°C, spin down briefly.
- Remove the comb, flush pockets with 1x MOPS running buffer.
- Arrange gel chambers inside the electrophorator, fill the tank with 1 x MOPS running buffer.
- Load 5µL of prestained standard, add 40µg sample/lane.
Blotting
- Blotting steps:
- Prepare transfer buffer and store in freezer (100 mL 10 x transfer buffer + 200 mL Methanol + 700 mL dH2O).
- Equilibrate nitrocellulose membrane, sponge, and filter paper in 1 x transfer buffer.
- Create a transfer sandwich with the gel, membrane, and other materials.
- Check for air bubbles between the gel and the membrane.
- Relocate the sandwich to the transfer apparatus, add cooling pack, and fill with transfer buffer.
- Transfer for 1h at 100V.
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Description
A step-by-step guide to performing a Western blot, including membrane preparation, antibody incubation, and detection. This protocol involves washing, blocking, and incubating the membrane with primary antibodies to detect protein transfer.
