Dec 09, 2024
Western blotting using NuPAGE system
- Ashley Seifert1
- 1University of Kentucky
Protocol Citation: Ashley Seifert 2024. Western blotting using NuPAGE system. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3qxz1v25/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: December 06, 2024
Last Modified: December 09, 2024
Protocol Integer ID: 114544
Keywords: ASAPCRN
Disclaimer
Note that any protocol involving animals should be reviewed and approved by your Institutional Animal Care and Use Committee (IACUC) before use.
Abstract
This protocol describes the procedure for western blotting with the NuPAGE system used by the Seifert Lab.
It supplements the manufacturer's guide to the NuPAGE electrophoresis system: https://tools.thermofisher.com/content/sfs/manuals/nupage_tech_man.pdf
Materials
For sample preparation and electrophoresis:
- Quantified protein samples
- NuPAGE™ LDS Sample Buffer (4X)Invitrogen - Thermo FisherCatalog #NP0008 (stored at 4 °C )
- NuPAGE Sample Reducing Agent (10X)Thermo Fisher ScientificCatalog #NP0009 (stored at 4 °C )
- NuPAGE Antioxidant Thermo Fisher ScientificCatalog #NP0005 (stored at 4 °C )
- NuPAGE gel for electrophoresis (stored at 4 °C )
- NuPAGE™ MES SDS Running Buffer (20X)Thermo FisherCatalog #NP0002 (stored at Room temperature )
- NuPAGE™ MOPS SDS Running Buffer (20X)Thermo FisherCatalog #NP0001 (stored at Room temperature )
- Novex Sharp Pre-stained Protein StandardThermo Fisher ScientificCatalog #LC5800 (stored at -20 °C )
For transfer:
- iBlot™ Transfer Stack, PVDF, miniThermo FisherCatalog #IB401002
- Tris-Glycine transfer buffer (10X)
- ~1.5 L 1X Tris-Glycine transfer buffer with 20% methanol
Protocol materials
NuPAGE Antioxidant Thermo Fisher ScientificCatalog #NP0005
Novex Sharp Pre-stained Protein StandardThermo Fisher ScientificCatalog #LC5800
iBlot™ Transfer Stack, PVDF, miniThermo FisherCatalog #IB401002
NuPAGE Sample Reducing Agent (10X)Thermo Fisher ScientificCatalog #NP0009
NuPAGE™ LDS Sample Buffer (4X)Invitrogen - Thermo FisherCatalog #NP0008
NuPAGE Antioxidant Thermo Fisher ScientificCatalog #NP0005
NuPAGE™ MOPS SDS Running Buffer (20X)Thermo FisherCatalog #NP0001
Novex Sharp Pre-stained Protein StandardThermo Fisher ScientificCatalog #LC5800
NuPAGE™ MES SDS Running Buffer (20X)Thermo FisherCatalog #NP0002
iBlot™ Transfer Stack, PVDF, miniThermo FisherCatalog #IB401002
Sample Preparation and Electrophoresis
Sample Preparation and Electrophoresis
Prepare samples On ice in individual 0.5 mL tubes according to the following chart:
| A | B | C | D | E | |
| Reagent | 8 wells | 10 wells | 12 wells | 15 wells | |
| Protein Extract (maximum) | 40 μg | 40 μg | 30 μg | 30 μg | |
| ddH2O | up to 19.50 μL | up to 16.25 μL | up to 13.00 μL | up to 9.75 μL | |
| 4X LDS Sample Buffer | 7.50 μL | 6.25 μL | 5.00 μL | 3.75 μL | |
| 10X Reducing Agent | 3.00 μL | 2.50 μL | 2.00 μL | 1.50 μL | |
| Total Volume (maximum) | 30.0 μL | 25.0 μL | 20.0 μL | 15.0 μL |
Note
- Maximum amount of protein and volume depends on size of wells
- Remember to keep a well open for a standard
- Any wells without samples should be filled with loading buffer to maintain an equal charge across the gel
- Be sure to keep the samples on ice to minimize degradation!
Incubate prepared samples for 00:10:00 at 70 °C to denature proteins and bind SDS
While samples are incubating, prepare the gel and the electrophoresis apparatus
Make 800 mL of running buffer by diluting 40 mL stock buffer with 760 mL ddH2O
Note
The use of MES or MOPS depends on antigen resolution desired. Refer to the NuPAGE migration guide: https://www.thermofisher.com/us/en/home/technical-resources/research-tools/image-gallery/image-gallery-detail.3436.html
Choose desired gel and open package
Note
- Wear gloves, as the buffer in the package contains sodium azide
- The gel percentage and matrix depend on the antigen resolution desired. Refer to the NuPAGE migration guide: https://www.thermofisher.com/us/en/home/technical-resources/research-tools/image-gallery/image-gallery-detail.3436.html
Rinse the gel cassette with ddH2O
Remove the comb from the top and the strip of tape from the bottom
Place the gel cassette into the apparatus and clamp shut
Add 1X running buffer to the middle chamber, ensuring that buffer covers the wells, and then to the outer chamber
Add 500 µL of NuPAGE Antioxidant Thermo Fisher ScientificCatalog #NP0005 to the inner chamber
Load 15 µL of sample into individual wells using gel loading pipet tips
Add 10 µL Novex Sharp Pre-stained Protein StandardThermo Fisher ScientificCatalog #LC5800 to one well
Attach the top of the electrophoresis apparatus, plug the leads into the power supply and turn the power supply on
Run at a constant voltage of 180 V until the blue dye front has reached the bottom of the gel
Note
- the yellow front is ~5 kDa
- run time is typically about 00:45:00 for MES and about 01:00:00 for MOPS
Transfer
Transfer
Remove the gel cassette from the apparatus and place onto paper towels. (The cassette is two pieces of plastic that enclose the gel.)
Using the provided metal tool, break the cassette at all edges by leveraging the tool between the plastic pieces
Making sure not to tear the gel, carefully separate the plastic pieces so that the gel remains on one
Using two hands, carefully pick up the gel, separating it from the remaining plastic piece, and place into ddH2O
Use the metal tool to cut off the wells and the bottom of the gel
Optional: The gel can be stained with Coomassie blue to visualize all protein
Activate the PVDF membrane in 100% methanol
Assemble the iBlot™ Transfer Stack, PVDF, miniThermo FisherCatalog #IB401002 in 1X Tris-Glycine transfer buffer containing 20% methanol in the following order: two filter papers, gel, PVDF membrane, two filter papers, sponge.
Note
Be sure to orient the gel onto the membrane in a logical manner. (i.e. keep the largest protein of the ladder on upper left). Use a plastic roller to roll out any bubbles after each stage of the transfer stack has been added.
Close and insert the cassette into the transfer box and close the lid, connecting the red lead to the red side and the black to black
Run at 75 V for 02:00:00 or at 55 V for 04:00:00 for higher molecular weight proteins
When finished, open the lid, remove the cassette, and carefully remove the sponge, filter papers and gel from the membrane, checking that the ladder has transferred from the gel to the membrane. Using forceps, quickly remove the membrane and place in a tray with 1X TBST
Optional: The membrane can be stained with Ponceau S to visualize all protein