Aug 29, 2025
Western Blot
- Kasandra Scholz1,
- Mary Gannon1,
- Talene Yacoubian1
- 1University of Alabama at Birmingham
Protocol Citation: Kasandra Scholz, Mary Gannon, Talene Yacoubian 2025. Western Blot . protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1195kvr2/v1
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: August 26, 2025
Last Modified: August 29, 2025
Protocol Integer ID: 225521
Keywords: ASAPCRN, detecting specific protein, protein analysis, standard western blot, reliable approach for protein analysis, western blot, specific antibody, immunoblot, proteins by molecular weight, separating protein, verification of molecular weight, selective detection
Abstract
This protocol describes the standard western blot (immunoblot) technique for detecting specific proteins in a complex sample. The procedure involves separating proteins by molecular weight using SDS-PAGE, transferring them to a membrane, and probing with target-specific antibodies to achieve selective detection. The method provides a reliable approach for protein analysis, including quantification of expression levels and verification of molecular weight.
Guidelines
DTT recipe: 0.25 M Tris-HCl pH 6.8, 200mM DTT, 8% SDS, 30% glycerol, 2mg bromophenol blue
Materials
- BCA protein assay and standards ready concentrations referenced: 2000, 1500, 750, 500, 250, 125 and 0 µg/µL
- PBS
- Kit buffers (prepare mixed at 1:50 ratio; add 100 µL to samples)
- Dryboil plate (for boiling samples)
- Heating block or dry plate capable of 95°C
- 15% acrylamide SDS-PAGE gels (cassettes)
- Running buffer
- Transfer buffer
- Nitrocellulose membrane
- Transfer sandwich materials: 2 sponges, 1 paper filter (per side), gel, nitrocellulose membrane
- Tweezers (for transfers)
- Containers for transfer at 100 V (power supply)
- PBS for rinsing and fixation
- 0.4% PFA in PBS (for fixation)
- TBST (Tris-buffered saline with Tween)
- 5% milk (blocking solution)
- Primary antibodies: BD anti-asyn (Cat# 610787; mouse) at 1:1000; GAPDH (Cat# 14C10; CS rabbit) at 1:10,000
- Secondary antibodies: anti-mouse HRP and anti-rabbit HRP, each used at 1:2000 in 5% milk
- ECL solution (prepare 1:1 mixture; ~2 mL per blot)
- Chemidoc (or equivalent imaging system)
- Kimwipes or absorbent paper
- Sheet protector (to place membranes on before applying ECL)
Troubleshooting
Safety warnings
- Listen out for popping of caps when boiling samples and do a quick spindown afterwards.
- Do not ever touch filters directly, use tweezers for transfers.
- Always check to make sure the buffer is bubbling/current is flowing during electrophoresis and transfer.
- Roll out any bubbles when assembling the transfer sandwich; ensure no bubbles remain between gel and membrane.
- Note about antibody reuse: Can save Ab! Store in a conical tube in the freezer. Can be used up to 3–4 times before Ab starts to wear off.
- During last wash before secondary incubation: prepare secondary antibody and let it mix on shaker until ready.
Before start
- Ensure ready-to-use BCA standard concentrations are prepared: 2000, 1500, 750, 500, 250, 125 and 0 µg/µL. add 5 µL of standards (no PBS) in triplicate in set wells. Dilute samples at 1:5 (4 µL PBS : 1 µL sample) in triplicate when needed, then add 100 µL of BCA kit buffers mixed at 1:50 ratio.
- For sample prep: target 20 µg protein in 10 µL sample;
- Prepare gels, running buffer, transfer buffer, nitrocellulose membranes, and transfer sandwich components in advance.
- Prepare blocking solution (5% milk) and TBST; pre-chill primary antibody mix if incubating overnight at 4°C.
Day 1
Prep samples for BCA assay: add in 5 µL of standards, no PBS. Ready-to-use concentrations should already be made up (2000, 1500, 750, 500, 250, 125 and 0 µg/µL). dilute samples at 1:5 (so 4 µL PBS : 1 µL sample), then add 100 µL of the kit buffers mixed at a 1:50 ratio.
make samples: 20 µg protein in 10 µL volume with 1XDTT for denaturing.
Day 2
Boil samples at 95°C for 10 minutes in a dryboil plate listen out for popping of caps, do a quick spindown afterwards.
Load samples (15% acrylamide gels) and run gel in running buffer put gels in cassettes facing inwards. Make note of which samples align with which well in the gels
Run at 80 V until samples leave the stacking layer, then run at 100 V until samples are at the bottom of the gel. Always check to make sure the buffer is bubbling/current is flowing.
Transfer samples in transfer buffer onto a nitrocellulose membrane at 100 V for 1 h.
Assemble transfer sandwich in the following order (from bottom to top):
Black side of comb on bottom
2 sponges
1 paper filter
Gel
Nitrocellulose membrane
1 paper filter
2 sponges
Roll out any bubbles! do not ever touch filter directly, use tweezers for transfers.
When transfer is done, isolate membrane.
Rinse blots in PBS, fix in 0.4% PFA for 30 minutes, then wash with TBST (3 quick rinses).
Block membranes in 5% milk for 60 minutes at room temperature on a shaker.
Incubate membranes in primary antibody overnight at 4°C in 5% milk.
can save Ab! Store in a conical tube in the freezer. Can be used up to 3-4 times before Ab starts to wear off.
7.3.25
Wash blot in TBST 3 x 5 minutes. during last wash, make 2º Ab and let mix on shaker until ready.
Incubate in secondary antibody for 1 hour at room temperature with shaking.
Wash blots 3 x 5 minutes in TBST.
Prepare ECL solution (1:1 mixture, ~2 mL per blot) and image on the Chemidoc.
Place membranes on a sheet protector and apply ECL solution ALL over the blot.
Incubate for 2 min in ECL and make sure there are no bubbles.
Make a pile of Kimwipes and lay them on top of blots to absorb ECL before taking pictures on the Chemidoc.