Aug 04, 2018
Western Blot Protocol (Cell Lysate) and Creating Running Gel
- Tyler Wenzel1
- 1University of British Columbia Okanagan
External link: http://www.tylerjwenzel.com
Protocol Citation: Tyler Wenzel 2018. Western Blot Protocol (Cell Lysate) and Creating Running Gel. protocols.io https://dx.doi.org/10.17504/protocols.io.sdpea5n
Manuscript citation:
Pointer, C., Wenzel, T.J., Klegeris, A. (2018). Extracellular Cardiolipin Regulates Select Immune Functions of Microglia. In review.
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
I use this protocol to quantify proteins in all my western blot protocols. This protocol consistently develops.
Created: August 04, 2018
Last Modified: August 04, 2018
Protocol Integer ID: 14479
Keywords: Western blot, proteomics, protein, antibody, protocol, immunology, neuroimmunology, cell lysate, western blot protocol, basic western blot protocol, western blot, running gel, cell, gel
Abstract
A basic Western Blot protocol that has been optimized specifically for cell lysates.
Troubleshooting
Before start
You will need to optimize the weight of your running gel before you begin. Most proteins will be separated with 8%-12% acrylamide gels. If your protein runs off your gel, you will need to use a heavier gel. See step 12 for more information.
CELL PROTEIN PREPARATION PROCEDURE
Seed 2mL of cells at desired density in 6mL TC-coated petri dish
Note: Attached to this step is a document that has all the solutions described in this protocol.
western blot 2017.docx Drug and stimulate cells as desired
Wash cells with ice-cold PBS
Aspirate the PBS and add 0.3mL ice-cold RIPA buffer
Incubate 5 min on ice
Scrape cells off the dish using a cold plastic cell scraper and transfer to a 0.5mL microcentrifuge tube
Centrifuge the samples at 10000xg for 5 minutes
Pipette the supernatant (containing the protein) into a fresh 1.5mL centrifuge tube, discard the pellet
GEL PREPARATION PROCEDURE (DAY 1)
Clean gel plates with distilled water (make sure they are “1.0mm” plates)
Place plates into green casting frame
Place casting frame (with plates) into the casting stand
Make lower gel.
If using a bigger protein, use a lower gel mix percentage
Guidelines for Acrylamide % in Gels
<60kDa = 12%
60-110 kDa = 10%
>110 kDa = 8%
IMPORTANT: Do NOT add TEMED until you’re ready to start pouring the gel, as it will begin gel solidification
Have gel components (minus the TEMED) and ethanol ready
Add TEMED to gel components and immediately pour the lower gel between the plates
Add 100uL-1mL ethanol over top of the lower gel until it is even
Let the lower gel sit for 20 minutes to solidify.
During this time you can:
- Make 1x Laemelli Running Buffer
- Make 1x Towbin Transfer Buffer
- Make upper gel
- Get plastic lane combs (make sure they are 1.0mm)
Pour out ethanol above lower gel
Wash lower gel with distilled water once
Pour out distilled water above lower gel
Remove excess distilled water using the corner of a piece of Whatman paper (do NOT touch the gel)
Add TEMED to the upper gel components and immediately pour the upper gel overtop the lower gel until overflowing
Immediately add lane combs to the stacking gel, making sure there are no bubbles
Let upper gel sit for 10 minutes to solidify
- During this time you can:
- Boil ladder (11uL Biotin ladder mixed with 11uL Opti Marker ladder) in water bath for 2 minutes – hold tube with tongs
- Boil proteins in heating block containing water at 90oC for 4 minutes
Carefully remove lane combs from the upper gel
Wash once with distilled water by causing the water to overflow out of the casting frame
RUNNING WESTERN BLOT PROCEDURE (DAY 1):
Move gel plates to a clamping frame and place in a gel running box
Add 1x Laemmli running buffer just below the top of the plates, and to the appropriate line in the gel box
Make sure there are no bubbles where the gels are by aspirating the bubbles off
Add 10uL of sample to each gel lane, by slowly releasing sample until the first pipette stop (you will need to run a standard curve of various volumes to determine which volume to use; you want to make sure you can detect higher and lower protein concentrations and do not saturate the bands)
Run the gel at 160V (3.00A) at room temperature until the dye front begins to run off the bottom (usually 1h20min)
While gel is running:
- Cut and label nitrocellulose membranes
- Cut Whatman paper (if necessary)
- Wash sponges in distilled water
- Make 1x Towbin transfer buffer
- Get a plastic tray to work in
- Label transfer boxes
WESTERN BLOT TRANSFER AND QUANTIFICATION PROCEDURE (DAY 1)
Once gel is complete, make your gel sandwiches for protein transfer onto the nitrocellulose membranes
Gel sandwich (from black side to clear side)
- Black sponge
- White Sponge
- 1 Piece of Whatman paper
- Gel
- Nitrocellulose membrane (with writing face down against the gel)
- 1 piece of Whatman paper
- Roll out air bubbles gently (use a roller, 50mL conical w/o lid, etc.)
- 2 pieces of Whatman paper
- White sponge
- Black sponge
- Roll out air bubbles firmly (use a roller, 50mL conical w/o lid, etc.)
- Seal gel sandwich and place in transfer box (black side of sandwich chamber should face black side of transfer box)
- Add an ice pack to the transfer box
- Fill area around transfer box with packed ice
- Fill transfer box with 1x Towbin transfer buffer
Run protein transfer at a current of 0.30A for 1h 15min (you will need to set a higher voltage but press run when current is selected)
Meanwhile, you can make 10mL Blocking Solution and store at 4oC (5% w/v Skim Milk Powder in TBS-T)
After protein transfer, move the membrane to a small membrane Tupperware box
Ponceau stain the nitrocellulose membrane to visualize transferred proteins and warm up camera
Take a picture of the stained transferred proteins with a 18-70mm lens
Wash membranes 3 times on a shaker with TBS-T for 5 minutes to remove Ponceau stain in shaker. Ensure there is no color left.
Add Blocking Solution to the membrane Tupperware box and shake for 1h
Pour off excess Blocking Solution
Add on 1o antibody dissolved in blocking solution (usually 1:1000 dilution) into membrane Tupperware box and shake overnight at 4o C. STORE EMPTY CONICALS IN FRIDGE OVERNIGHT as you can use the 1o antibody for months.
Note: You can add the 1o antibody for only an hour at room temperature, but your bands will be less clear and there may be more background bands.
WESTERN BLOT TRANSFER AND QUANTIFICATION PROCEDURE (DAY 2)
Place 1o antibodies back in the stored 50mL conical and freeze for reuse
Wash membrane with TBS-T at room temperature for 1h on a shaker (add TBS-T every 10 minutes)
Add on 2o antibody dissolved in blocking solution into membrane Tupperware box and shake at room temperature for 1h
Wash membrane with TBS-T at room temperature for 1h on a shaker (add TBS-T every 10 minutes) and turn on chemiluminescent camera at this time
Develop and Quantify Blots
Dry off membrane in Saran wrap and place in a new Tupperware container
Make ECL solution
Add 1mL light ECL reagents (per membrane) and 1mL dark ECL reagent (per membrane) into a 15mL conical
Important: Change tips to avoid contaminating ECL reagents)
With a 1mL pipette, add 2mL ECL solution over the membrane drop by drop until covered
Cover the membrane box (with aluminum foil) for 5 minutes
Take picture using chemiluminescent machine in the anteroom
Take picture at 70mm using the 18-70mm lens, using the aperture ring to get the correct exposure
OR the settings that are specific to the chemiluminescent machine available in your lab.