Apr 29, 2026
Western Blot
- Sriparna Mukherjee1,2,3,4,
- Amandine EVEN1,2,3,5,4,
- Louis-Éric Trudeau1,6,2,3,5,4
- 1Department of pharmacology and physiology, Faculty of Medicine, Université de Montréal;
- 2Neural Signaling and Circuitry research group (SNC);
- 3Center for Interdisciplinary Research on the Brain and Learning (CIRCA);
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 5Institut Courtois d’innovation biomédicale;
- 6Department of neuroscience and physiology, Faculty of Medicine, Université de Montréal
Protocol Citation: Sriparna Mukherjee, Amandine EVEN, Louis-Éric Trudeau 2026. Western Blot. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7okwqvwz/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: April 20, 2026
Last Modified: April 29, 2026
Protocol Integer ID: 315407
Keywords: western blot this protocol, western blot, immunoblotting, chemiluminescent imaging, transfer to nitrocellulose, sd
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000525
Abstract
This protocol describes SDS-PAGE, transfer to nitrocellulose, immunoblotting, chemiluminescent imaging, stripping, reprobing, and densitometric analysis.
Materials
**Gel electrophoresis**
1. SDS-PAGE gels (8-12%)
- Resolving gel buffer 1X 1.5 Molarity (M) Tris 8.8 (Biorad Cat#1610798)
- Stacking gel buffer 1X 0.5 Molarity (M) Tris-HCl 6.8 (Biorad Cat#1610799)
- 30% Acrylamide/Bis Solution, 29:1 (Biorad Cat#1610156)
- TEMED (Bioshop Canada Cat#TEM001.25)
- SODIUM DODECYL SULFATE (SDS) 100mg/mL from powder (Bioshop Canada Cat#SDS001.100)
- APS 100 mg/mL from powder
2. Electrophoresis Buffer 1X - 100 mL EB buffer 10X (Biorad Cat# 1610732EDU) + 900 mL MilliQ Water
**Transfer**
1. Nitrocellulose membranes, sponges and transfer buffer (Biorad Cat#1704271)
2.Transfer system (semi-dry) or TransBlot Turbo Transfer System (Biorad)
**Blocking and antibody incubation**
1. Every Blot Blocking Buffer (BioRad, Cat# 12010020)
2. TBST Buffer - 100 mL Tris-buffered saline 10X (Biorad Cat#1706435EDU) + 900 mL MilliQ water + 1 mL Tween-20
3. TBST-BSA - 50 mL TBST 1X + 0.5 g Bovine Serum Albumin (Sigma, Cat# A7906)
**Detection of signal**
1. Clarity Western ECL Substrate (Bio-Rad, Cat# 1705061)
2. Imaging System - ChemiDoc™ MP (Bio-Rad)
3. Image Analysis Software - ImageJ (NIH)
**Antibodies**
1. ZO-1 (Rabbit, 1:1000) - Cell signaling technology, Cat# 13663, RRID: AB_2798287
2. ZO-2 (Rabbit, 1:1000) - Cell signaling technology, Cat# 2847, RRID: AB_2203575
3. GFAP (Rabbit, 1:2000) - Agilent Technologies Singapore, Z0334
4. IBA1 (Rabbit, 1:1000) - Fujifilm Wako Pure Chemical Corporation, Cat# 016-20001, RRID: AB_839506
5. Beta actin peroxidase (Mouse, 1:15000) - Sigma, Cat# A3854, RRID: AB_262011
6. DAT (Rat, 1:1000) - Millipore, Cat# MAB369, RRID: AB_2190413
7. Claudin 1 (Rabbit, 1:1000) - Abcam, Cat# ab211737, RRID: AB_3082989
8. TH (Mouse, 1:1000) - Millipore, Cat# MAB318, RRID: AB_2201528
9. Peroxidase-conjugated AffiniPure Donkey Anti-Rabbit IgG (Donkey, 1:5000) - Jackson ImmunoResearch Laboratories, 711-035-152, RRID: AB_10015282
10. Peroxidase-AffiniPure Goat Anti-Mouse IgG (Goat, 1:5000) - Jackson ImmunoResearch Laboratories, 115-035-146, RRID: AB_2307392
11. Peroxidase-AffiniPure Goat Anti-Rat IgG (Goat, 1:5000) - Jackson ImmunoResearch Laboratories, 112-035-003, RRID: AB_2338128
Before start
Confirm that the ChemiDoc system, transfer apparatus, and antibodies are functional and available before loading gels.
Gel electrophoresis
4h 55m 40s
Separate proteins on 8–12% SDS-PAGE gels.
Select the gel percentage according to the target molecular weight and maintain identical gel conditions for directly compared groups.
Percent of polyacrylamide to select depending the size of the target
2h
Prepare the resolving gel:
Mix the reagents in a test tube according to the table below and the chosen percentage.
Dosages of reagent to prepare one gel
Vortex for 00:00:20 .
20s
Pour the gel and quickly add 1 mL of methanol to the gel.
Ensure it does not leak.
Allow to polymerize for a minimum of 00:20:00 .
Prepare the stacking gel during this time
20m
Prepare the Stacking gel (4% of Polyacrylamide)
Mix everything except the TEMED and APS.
Dosages of reagent to prepare two gels
When polymerization of the resolving gel is completed, empty the methanol and wipe with a KimeWipe without touching the gel.
Add the APS and TEMED in the mix for the stacking gel, and vortex for 00:00:20 .
20s
Add the stacking gel on the top of the resolving gel, until it reaches the top of the glass.
Directly insert the combs and wipe away any excess.
Allow to polymerize for a minimum of 00:20:00 .
20m
Mix each sample to obtain 20 µg total protein per lane. Add sample buffer containing the reducing reagent.
15m
When the gel is ready, run the SDS-PAGE:
Fill the tank with the Electrophoresis Buffer (EB) 1X
Gently remove the combs and load the scale and the sample.
Try to use a single pipette for the entire loading process to avoid having two different levels of precision between samples.
Select a voltage of 75 volts and run for approximately 00:30:00 for stacking.
30m
When you see the scale well separated, increase to a maximum of 100 volts from 01:00:00 to 01:30:00 for resolving.
When the migration is completed, the transfert has to be done within 15 minutes
1h 30m
Transfer
10m
Freshly prepare 100 mL of Transfer Buffer (TB) 1X mixing 20 mL Transblot Turbo 5X and 80 mL MilliQ Water (if TransBlot Turbo Transfer System used)
Transfer resolved proteins onto nitrocellulose membrane using a semi-dry transfer apparatus or TransBlot Turbo Transfer System (Biorad).
Wash the plates with Milli-Q water to remove traces of EB and collect the gels in the TB.
Prepare the sandwich in the cassette of the transfert machine in the following order:
- a TB-soaked sponge
- a nitrocellulose membrane
- the gel
- another TB-soaked sponge.
Compact this sandwich using a roller. Ensure the layers are not wrinkled, without stretching the gel. Be careful with bubbles.
Close the cassette and start the transfer.
If the machine TransBlot Turbo Transfer System is used, chose this sequence in the settings
List -> Bio-Rad -> 2 mini or 1 midi -> 1.5mm Gel 2.5A – 25V – 10min -> Run -> Run A or Run B (depending the cassette)
Let the transfert during 00:10:00 .
10m
Verification of the transfert quality
30s
Put the membrane in the Ponceau solution 00:00:30 with gentle agitation.
30s
Rince 2-3 times with milliQ water with gentle agitation.
Bands reflects the presence of proteins and confirms that the transfer was successful.
If membrane cuts are needed to separate samples based on molecular weights, now is the time to perform them.
Wash with TB once and 2 times with milliQ water.
Immunodetection
2h 40m
Block membrane in 10 mL of EveryBlot blocking buffer for 00:05:00 at Room temperature with gentle shaking.
5m
Dilute the appropriate primary antibody (as listed above) in TBST-BSA solution.
Remove the blocking solution and incubate the membrane with the diluted primary antibody , overnight at 4 °C with gentle shaking.
Wash membrane 5 times in TBST for 00:05:00 each with shaking.
25m
Dilute the HRP-conjugated secondary antibody (as listed above) in TBST-BSA solution.
The β-actin primary is already coupled with HRP and doen't need the secondary step.
Incubate membranes with the matching secondary antibody for 01:30:00 at Room temperature with gentle shaking.
1h 30m
Wash membrane 5 times in TBST for 00:05:00 each with shaking
25m
Develop blot:
10m
Prepare Clarity Western ECL substrate with 500 µL of peroxidase for 500 µL of enhancer.
Vortex thoroughly before use!
β-actin gives a very strong signal and the ECL substrate needs to be diluted with 100 µL of peroxidase, 100 µL enhancer and 300 µL TBST 1X , using 500 µL per blot and reusable blot for 3 blots.
Apply 1 mL of Clarity Western ECL substrate per blot.
1 mL can be reusable for 3 blots
5m
Wait up to 00:05:00 of incubation before capturing images, except for β-actin which must be captured directly to avoid saturation.
Capture images with a ChemiDoc MP system.
Acquire multiple exposures if necessary to avoid signal saturation.
Strip and reprobe
45m
If necessary, the membrane can be striped and reprobed, for example for the β-actin as the loading control if it was in the same molecular weight than another target.
Stripping:
Add 5 mL of stripping solution to the membrane and incubate 00:30:00 under fume hood with gentle agitation.
30m
Rinse 3 times with 5 mL of MilliQ water for 00:05:00 with agitation.
15m
Redo steps 12 to 19.
Data analysis
Open image in ImageJ.
Use the same analysis workflow for all membranes in a comparison set.
Quantify the integrated density of the target band and the corresponding β-actin band for each sample.
Normalize data in Excel.
Divide target intensity by β-actin intensity for each lane.
Export normalized values for statistical analysis in GraphPad Prism or equivalent software.
Troubleshooting
Weak target signal: Increase exposure, verify transfer quality, and optimize antibody incubation using pilot blots.
High background: Increase wash stringency or reduce antibody concentration.
Uneven bands across lanes: Re-check protein normalization and transfer setup.
β-actin signal inconsistent: Use fresh samples or run a parallel loading control blot if stripping affected signal.