Aug 09, 2024
Immunoblotting using precast gels
- Francisco Bustos1,2
- 1Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD, USA;
- 2Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD, USA
Protocol Citation: Francisco Bustos 2024. Immunoblotting using precast gels. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6j8pl5d/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 08, 2024
Last Modified: August 09, 2024
Protocol Integer ID: 104903
Abstract
Immunoblotting is a key technique to visualize changes in protein levels upon treatments. This technique can be challenging and established procedures are required to ensure reproducibility. Here we present our optimized protocol for immunoblotting of protein samples using precast gels and semiwet transfer. This protocol can be used to analyze samples from cell extracts and from in vitro reactions.
Protocol materials
PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDaThermo FisherCatalog #26620
PowerPac™ HC Power SupplyBio-Rad LaboratoriesCatalog #1645052
NUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007
NuPAGE Sample Reducing Agent (10X)Thermo Fisher ScientificCatalog #NP0009
Gel KnifeThermo FisherCatalog #EI9010
XCell SureLock® Mini-Cell and XCell IITM Blot Module KitInvitrogen - Thermo FisherCatalog #EI0002
SNAP id® 2.0 Blot RollerMerck MilliporeSigma (Sigma-Aldrich)Catalog #SNAP2RL
PowerPac™ HC Power SupplyBio-Rad LaboratoriesCatalog #1645052
Grade 3MM Chr Blotting Paper, sheet, 46 × 57 cmCytivaCatalog #3030-917
Carnation Instant Non Fat Dry MilkAmazon.comCatalog #12428935
Immobilon-P PVDF Membrane, 0.45um, rollMerck MilliporeSigma (Sigma-Aldrich)Catalog #IPVH00010
Amersham™ Protran® Western blotting membranes nitrocelluloseMerckCatalog #GE10600041
Sponge Pad for BlottingThermo FisherCatalog #EI9052
Ponceau S solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #Ponceau S solution
Fisher BioReagents™ Bovine Serum Albumin Heat Shock TreatedFisher ScientificCatalog #BP1600-100
GlycineFisher ScientificCatalog #BP381-500
Sodium dodecyl sulfate Merck MilliporeSigma (Sigma-Aldrich)Catalog #62862
Polysorbate 20Thermo Fisher ScientificCatalog #233360010
Methanol, 99.9%, for analysisThermo Fisher ScientificCatalog #176840025
MES-SDS Buffer (20X)Boston BioproductsCatalog #BP-177
Polysorbate 20Thermo Fisher ScientificCatalog #233360010
NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 12-wellThermo FisherCatalog #NP0322BOX
Tris BaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #648310
GlycineFisher ScientificCatalog #BP381-500
Western blot boxes, 3 1/2 x 2 9/16 x 1in. 8.9 x 6.5 x 2.5cmFisher ScientificCatalog #NC1126730
Safety warnings
All steps must be performed using personal protective equipment including gloves and eye protection.
Sample preparation
Sample preparation
Mix NUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007 and NuPAGE Sample Reducing Agent (10X)Thermo Fisher ScientificCatalog #NP0009 in a 7:3 ratio (for example: 70 µL LDS sample buffer and 30 µL of Reducing Agent).
In a 1.5 mL microcentrifuge tube add the following reagents in order: LDS sample buffer/Reducing Agent mix, IPMS lysis buffer, protein sample. For example.
| Sample volume | LDS sample buffer/Reducing Agent | |
| 10 or 15 microliters | 5 microliters | |
| 20 | 7 | |
| 30 | 10 |
Poke hole in top of 1.5 mL microcentrifuge tube with a hypodermic needle.
Boil samples at 95 °C for 00:05:00 using a ThermoMixer F1.5.
Equipment
Eppendorf ThermoMixer F1.5
NAME
ThermoMixer
TYPE
Eppendorf
BRAND
5384000020
SKU
LINK
5m
Centrifuge for 00:00:10 in a Mini Centrifuge at Room temperature
Equipment
Fisherbrand Mini-Centrifuge
NAME
Centrifuge
TYPE
Fisherbrand
BRAND
12-006-901
SKU
LINK
10s
Samples can be stored at -20 °C until the day of running SDS-PAGE electrophoresis.
SDS-PAGE Electrophoresis
SDS-PAGE Electrophoresis
Prepare 500 mL 1X MES-SDS Buffer (50 millimolar (mM) MES, 50 millimolar (mM) Tris Base, 0.1 Mass / % volume SDS, 1 Mass / % volume EDTA, pH 7.3) by diluting MES-SDS Buffer (20X)Boston BioproductsCatalog #BP-177 stock 1:20 in Milli-Q water.
Unpack a NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 12-wellThermo FisherCatalog #NP0322BOX and remove protective tape and comb.
Assemble gel running tank XCell SureLock® Mini-Cell and XCell IITM Blot Module KitInvitrogen - Thermo FisherCatalog #EI0002 by placing gel in the tank in front of the buffer core and a second gel or a buffer dam behind the
buffer core. Lock the gel tension wedge in place. Add 1X MES-SDS buffer between the gels and outside the buffer core.
Load 8 µL protein molecular weight marker PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDaThermo FisherCatalog #26620 . If needed load 2 µL at the end of the gel.
Load samples to a maximum volume of 20 µL .
Place gel tank lid and connect to power supply PowerPac™ HC Power SupplyBio-Rad LaboratoriesCatalog #1645052 matching colors (for positive and negative).
Run gel at 150 V for 02:00:00 at Room temperature or as needed depending on the protein molecular weight and desired separation.
2h
Transfer
Transfer
Prepare 12X transfer buffer:
- 58 g Tris base Tris BaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #648310
- 190 glycine GlycineFisher ScientificCatalog #BP381-500
- Milli-Q water up to 2 L
Prepare 2.5 L of 1X transfer buffer (48 millimolar (mM) Tris, 39 millimolar (mM) Glycine, 20 % (v/v) Methanol) by mixing 212 mL 12X Transfer buffer with 1788 mL milli-Q water and 500 mL MethanolMethanol, 99.9%, for analysisThermo Fisher ScientificCatalog #176840025 .
Cut Immobilon-P PVDF Membrane, 0.45um, rollMerck MilliporeSigma (Sigma-Aldrich)Catalog #IPVH00010 or Amersham™ Protran® Western blotting membranes nitrocelluloseMerckCatalog #GE10600041 membrane to the required gel size (usually 9x7 cm) and activate PVDF membrane by incubating in methanol for at least 00:01:00 .
Note
Do not put Nitrocellulose membrane in methanol
1m
Cut Grade 3MM Chr Blotting Paper, sheet, 46 × 57 cmCytivaCatalog #3030-917 to the required gel size (usually 9x7 cm).
Prepare Sponge Pad for BlottingThermo FisherCatalog #EI9052 . If dirty, boil in warm tap water in the microwave for 00:05:00 . Soak sponge pads in 1X transfer buffer in a plastic tray.
5m
Remove the gel from the cassette by separating the cassette plates with a Gel KnifeThermo FisherCatalog #EI9010 . Using the gel knife, cut off the bottom part and wells of the gel as needed.
Place gel in a plastic tray with 1X transfer buffer.
Make transfer sandwich inside the cathode core of the blot module in the following order:
- 2 sponge Pad
- 1 filter paper + gel (grab this from transfer buffer tray), Remove air bubbles by carefully rolling a SNAP id® 2.0 Blot RollerMerck MilliporeSigma (Sigma-Aldrich)Catalog #SNAP2RL on top of the gel.
- 1 presoaked PVDF or Nitrocellulose membrane. Remove air bubbles using the Blot Roller.
- 1 filter paper. Remove air bubbles using the Blot Roller.
- Sponges up to fill cavity (6-7 total)
Close blot module with the anode core and place this in the buffer chamber. Lock the blot module using the gel tension wedge.
Place lid and connect to power supply PowerPac™ HC Power SupplyBio-Rad LaboratoriesCatalog #1645052 matching colors (for positive and negative).
Run gel at 35 V for 01:30:00 at Room temperature .
1h 30m
Unlock blot module by releasing the tension wedge and remove from buffer chamber. Remove membrane from the sandwich and cut excess membrane using scissors.
Incubate membrane in Ponceau S solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #Ponceau S solution in a plastic tray for 00:05:00 with shaking in a rocking shaker.
Equipment
Rocking Shaker
NAME
Shaker
TYPE
Ohaus
BRAND
30391966
SKU
LINK
5m
Wash with milli-Q water 3 times, quick washes. Image in Chemidoc MP if needed.
Equipment
ChemiDoc™ MP Imaging System
NAME
Imaging System
TYPE
Bio-rad
BRAND
12003154
SKU
Blocking
Blocking
1h
1h
Make 1X TBS-T (Tris buffer saline-Tween 20: 20 millimolar (mM) Tris-HCl pH=7.4, 100 millimolar (mM) NaCl, 0.1 % (v/v) Tween-20).
For 1L:
- 20 mL 1 Molarity (M) Tris pH=7.4
- 20 mL 5 Molarity (M) NaCl
- 1 mL Tween-20 Polysorbate 20Thermo Fisher ScientificCatalog #233360010
- Milli-Q water up to 1 L
Make Blocking solution: 1X TBS-T 5% skimmed milk:
- 12.5 g powder skimmed milk Carnation Instant Non Fat Dry MilkAmazon.comCatalog #12428935 .
- Fill up to 250 mL with 1X TBS-T.
Add Blocking solution to a Western Blot box Western blot boxes, 3 1/2 x 2 9/16 x 1in. 8.9 x 6.5 x 2.5cmFisher ScientificCatalog #NC1126730 and place the membrane in this solution. Incubate in a rocking shaker for at least 01:00:00 at Room temperature
1h
Primary antibody
Primary antibody
1h
1h
Prepare antibody in 1X blocking solution (for HRP detection) or in TBST 5 Mass / % volume BSA Fisher BioReagents™ Bovine Serum Albumin Heat Shock TreatedFisher ScientificCatalog #BP1600-100 (for infrared detection) at the desired concentration.
Discard blocking solution from Western blot box and replace by diluted antibody. Incubate at 4 °C Overnight in rocking shaker.
20h
Next day recover antibody in tube and wash membrane 3 times for 00:05:00 with 1X TBS-T
5m
Secondary antibody
Secondary antibody
1h
1h
Prepare HRP or fluorescently labelled secondary antibody in blocking solution or TBST respectively.
Add diluted secondary antibody to Western blot box a incubate at Room temperature for 01:00:00 in rocking shaker.
1h
Wash membrane 4 times for 00:05:00 with 1X TBS-T
5m
Image in Chemidoc MP for HRP detection or LI-COR Odyssey FC imager for infrared detection.
Equipment
ChemiDoc™ MP Imaging System
NAME
Imaging System
TYPE
Bio-rad
BRAND
12003154
SKU
Equipment
Odyssey CLx
NAME
Imaging System
TYPE
LI-COR
BRAND
Odyssey CLx
SKU
LINK
Membrane can be stored up to a week at 4 °C until performing stripping.
Stripping
Stripping
10m
10m
Prepare Stripping buffer:
- weight 15 g glycine GlycineFisher ScientificCatalog #BP381-500
- Dissolve in 800 mL milli-Q water
- Adjust pH to 2.2
- add 1 g SDS Sodium dodecyl sulfate Merck MilliporeSigma (Sigma-Aldrich)Catalog #62862
- 10 mL Tween 20 Polysorbate 20Thermo Fisher ScientificCatalog #233360010
- Bring volume up to 1 L with milli-Q water
Incubate membrane 00:10:00 with stripping buffer in rocking shaker at Room temperature .
10m
Repeat step 37
Wash twice with TBST
go to step #28 and probe with a different antibody.