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College of Life Sciences
Microbiology and Molecular Biology

Western Blot Protocol

  1. Take harvested cells in RIPA buffer out of the freezer, thaw
  2. Sonicate 15 seconds at 10-12 cycle, centrifuge 10 min at max speed (performed in cold room)
  3. Put 4ul of supernatant into 36 ul of ddH2O and perform BCA.
  4. Use BCA values to determine the amount of supernatant to use, then q.s to 10uL with ddH2O for each sample in pcr tubes.
  5. Add 10uL of laemmli buffer solution to each sample and run through “western denature” protocol on PCR machine. Make sure the reaction volume is set to 20 uL (You have to make up the solution. See instructions below).
  6. Assemble cassettes, placing your gel on one side, openings facing in, and either another gel on the other side or a cassette buffer. Place in western blot container checking to make sure positive and negative nodes are properly oriented.
    1. Long electrodes on side with chords, short on the other side
  7. Fill cassette and container appropriately with 1x Running Buffer =
    1. Place ice packs in the container, tightly enough so they don’t float
    2. Fill cassettes all the way (wells should be facing in so that the running buffer fills the gel mold) 
    3. Fill Container to the indicated line
  8. Load the ladder (2uL) and protein samples (15 uL) into the gels
  9. Run at 50V for 1 hour (or until it runs off the stacking gel) and then turn it up to 100V for 1.5-3 hour. If it is slow, you may turn the voltage up to 200V as long as ice is constantly replaced.
    1. Follow this link for instructions if you get an E1 error
  10. WATCH it until the blue line is at the bottom of the gel and then STOP it (CAREFUL NOT to let it run off the gel). Depending on the molecular weight of the protein you are looking for you may need to run shorter or longer amount of time. Consult the ladder.
  11. Soak sponges, membranes, filter paper in 1x transfer buffer. (Check recipe to dilute 10x transfer buffer on Tessem protocol since it is not only H2O)
  12. Sandwich gel and membrane between 2 sponges and filter paper in the cassette. (gel to back) The order is as follows:
    1. Back side (black)
    2. Sponge 
    3. filter paper  
    4. gel
    5. membrane
    6. filter paper
    7. sponge
    8. front side (clear) 
      *Ensure that there are no bubbles by rolling with pipette tip.
  13. Put the cassette in black and red container with black side facing black side and white fastener facing up. Add stir bar and ice pack, then fill container with transfer buffer.
  14. Start run at 30V for 2 hours
  15. Remove membrane from cassette
  16. Briefly rinse in black box with DD water
  17. Block in sufficient Blocking Buffer(BB) to barely cover membrane for an hour or more in black box, on a shaker with lid on.
  18. Make up Primary Antibody in 10mL BB per membrane, use defined concentration for the antibody you are using (look online, frequently 1:1000 but must check for each antibody).
  19. Put it in the container on the shaker in the cold room overnight
  20. Save the primary antibody for future use. 
  21. Rinse 3x in .1% PBS-Tween for 5 min each on shaker with lid on 
  22. Make up secondary Antibody in 10mL BB per membrane, check concentrations, often 1:20,000 or more.
  23. Incubate in closed box on shaker at room temp 1 hour.
  24. Rinse membrane off with ddH2O.
  25. Dry in 2 pieces of filter paper in the dark (light sensitive) for 30 minutes.
  26. Image.

PBS-Tween

  • 900 mL PBS
  • 1.0 mL Tweene
  • q.s. 1L H2O

Laemmli Buffer Solution

  • 950uL 2x Laemmli
  • 50uL 2-mercaptoethanol

You can half or quarter this if only filling 18 samples.

Total Protein Stain

  1. Remove membrane from cassette following transfer step.
  2. Fully dry membrane on top of a piece of filter paper 40 to 60 minutes at room temperature, or overnight at room temperature.
  3. Rehydrate in PBS (no tween) for 5 minutes at room temperature on the shaker.
  4. Rinse membrane with ddH2O.
  5. Incubate membrane with 5 mL of Revert 700 Total Protein Stain solution for 5 minutes at room temperature on the shaker.
  6. Thoroughly decant Protein Stain.
  7. Rinse the membrane with 5 mL of Revert 700 Wash Solution two times for 30 seconds at room temperature with gentle shaking.
  8. Thoroughly decant Wash Solution.
  9. Rinse membrane with ddH2O.
  10. Immediately image the membrane on the LiCor machine in the 700 nm channel.
  11. Rinse with ddH2O
  12. Incubate membrane with 5 mL of Revert 700 Decant solution for 5 minutes.
  13. Rinse with ddH2O
  14. Resume Western at the blocking step.

Quantifying Western Protocol

  1. Image your westerns, save two separate copies for your target protein and your reference protein(Tubulin, etc)
  2. Open image up in FIJI, change image to 8-bit in “Image, type”
  3. Using the rectangular tool, draw a rectangle that covers the entire first column
  4. Press control one to set as standard size, then press control two to create a copy.
  5. Drag copied box over to the next column and repeat for each sample
  6. After the last sample is created, press control 3 to create a graph of each sample.
  7. Select the magic wand tool and select the area under the curve. If there are more than one peaks and the magic wand is picking up on that, use the line tool to make an additional line at the base of your desired peak.
  8. This will automatically calculate the area under the curve, copy and paste into an excel to calculate target protein divided by control protein.