Oct 14, 2020
Western Blot
This protocol is a draft, published without a DOI.
- 1Lankenau Institute for Medical Research
- LIMR
Protocol Citation: James D Montgomery 2020. Western Blot. protocols.io https://protocols.io/view/western-blot-bm6ik9ce
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 08, 2020
Last Modified: October 14, 2020
Protocol Integer ID: 42922
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Materials
MATERIALS
NuPAGE Antioxidant Thermo Fisher ScientificCatalog #NP0005
12-well NUPAGE 10% Tris-Bis gelThermo Fisher ScientificCatalog #NP0302BOX
MethanolFisher ScientificCatalog #A452-4
NuPAGE™ MOPS SDS Running Buffer (20X)Thermo FisherCatalog #NP0001
NuPAGE™ LDS Sample Buffer (4X)Thermo FisherCatalog #NP0007
NuPAGE™ Sample Reducing Agent (10X)Thermo FisherCatalog #NP0009
NuPAGE™ transfer buffer Thermo Fisher ScientificCatalog #NP0006
Gel Electrophoresis
Gel Electrophoresis
Make loading dye using LDS Sample Buffer (4X) and Reducing agent (10X). For 20 µL of sample, add 7.5 µL sample buffer and 3.5 µL reducing agent, keeping in mind that each well holds 30 µL total.
Combine sample and loading dye and heat at 95 °C for 00:05:00 .
Prepare precast gel by removing comb and the tape at the bottom of the gel chamber, making sure to remove any excess gel that could block the top of the wells. Construct the gel chamber in the gel tank such that the well openings of the precast are facing inwards toward eachother. Fill the gel chamber with 200 mL ((1X) MOPS running buffer and 500 µL antioxidant.
Remove samples from heat and cool On ice for 00:05:00 .
Spin samples at 10K for 00:05:00
Load samples into gel using hamilton pipette tips. Be sure to record the order of the samples in the gel noting that samples on the edges of the gel are more likely to become distorted. Placing the marker in the middle most gel will provide a more reliable size ladder for size estimation.
Run gel at 100V for 01:30:00 or until the protein has migrated far enough into the gel.
Transfer
Transfer
While the gel is running prepare 1 L Transfer Buffer using 850 mL DiH20 , 50 mL 10x Transfer Buffer , and 100 mL methanol and place in cold room for duration of gel run.
Soak membrane (for PVDF soak membrane in methanol, for nitrocellulose soak membrane in transfer buffer.) Do not touch membrane with ungloved hands.
Soak sandwhich components (2 sheets of filter paper and 2 pads) until completely damp. Make sandwhich being careful to avoid trapping any air between layers. Place cassette in transfer tank with transfer buffer and run for 1 hour.
Blocking
Blocking
Remove membrane from sandwhich, noting the orientation relative to the gel. Wash the membrane in 1X PBST for 00:05:00
Block membrane for 01:00:00 with 5% milk in PBST
Blot with desired Ab.