Jan 16, 2024

Public workspaceWestern Blot V.1

This protocol is a draft, published without a DOI.
  • 1University of California, San Diego
Yiqin Shen
Open access
Protocol Citation: Yiqin Shen 2024. Western Blot. protocols.io https://protocols.io/view/western-blot-c7fzzjp6
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: January 11, 2024
Last Modified: January 16, 2024
Protocol Integer ID: 93401
Abstract
This is a protocol of Leo Parra-Rivas, PhD.
Preparing Proteins
Preparing Proteins
Dilute protein samples to Concentration1.25 µg/µL . For running a 10-well gel, each loading sample should be around Amount15 µL to Amount20 µL .

Add 4x sample buffer to protein samples.
Denature protein at Temperature95-100 °C for Duration00:05:00 toDuration00:10:00

15m
Running the Gel
Running the Gel
Secure the gel (NuPAGE 4-12% Bis-Tris Gel; 1.5mm 10 wells) into the module. Fill the tank with running buffer. Load Amount15 µL of the ladder, and Amount20 µL of the samples (25ug/20ul, more if less concentrated).
Run at 70-80V for Duration01:15:00 . Until the blue band reaches the end of the gel, run for 10 more min.

1h 15m
Ensure the strip on the bottom is removed. Remove the comb without breaking the wells.
Running Buffer: 1900ml H2O, 100ml Running buffer 20X
You may add more buffer if the buffer level is low.
You may increase the voltage to 100V.
Transferring the Gel
Transferring the Gel
Assemble the module according to instructions (membrane = 0.2ul pores; gel needs to be flipped). Ensure all bubbles are removed.
Add transfer buffer (ice cold) to the tank. Securely locate the module. Add ice around the tank before running.
Run at 25-30V for Duration01:25:00

1h 25m
transfer buffer: 1500ml H2O, 400ml methanol, 100ml transfer buffer 20X
Staining
Staining
Fixation: rinse membrane with 1xPBS. Incubate in 0.4% PFA for Duration00:30:00 .

30m
Blocking: wash with 1xPBS, 3 times Duration00:10:00 . Block with 5-10% milk for Duration01:00:00 .

1h 10m
Primary antibody: Specific primary antibody diluted in 5% nonfat dried milk in TBST, Temperature4 °C overnight with rocker.
Wash: Recover the primary antibody, and rinse with wash buffer. Wash for Duration00:30:00 with wash buffer

30m
Wash buffer: 1800ml H2O, 200ml 10X PBS, 2ml Tween-20
Secondary antibody: Secondary antibody 1:2000 in milk, Duration01:00:00 TemperatureRoom temperature . Add approximately 10 ml to each tray.
Wash: Rinse and wash with wash buffer. The membrane can be stored in the wash buffer
1h
Imaging
Imaging
5m
Mix imaging reagent 1:1. Either apply reagent to membrane before imaging, or incubate the membrane in mixed reagent for Duration00:05:00 while rocking.
Avoid bubbles when placing the membrane on the machine.
Single image, start with 0.1s exposure and gradually increase until desired exposure is achieved.
More reagent can be added if signal is reduced.

5m