Questions and Answers
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.
False
The gel should be run at ___________ V through the resolving gel.
100V
What is the composition of the transfer buffer?
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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, _____________.
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How long should the transfer process last?
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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.
