Western blot protocol: Simplified steps to a deceptively detailed technique

1c2bca1 Amy Palubinsky

I remember it like it was yesterday. I was in a collaborator's lab learning a new protocol and needed to run a confirmatory Western blot before continuing. “No problem,” I said, “I’ve run thousands. Just point me in the right direction and I won’t bother you one bit.” Haha—come on, Amy—there are a million ways to run a Western blot, and every lab does it differently. 

In fact, because I know this to be true, I wrote a seven-page protocol for our lab to help streamline the Western blotting process. Here, I smush that protocol down to the length of a blog post by pointing out the essentials and some tips and tricks. This is not meant to be a comprehensive protocol, but more so a reference for newly inducted members of the Western blot society.

  • Choose the correct percentage gel. (Whether you’re making the gel on your own or using a precast gel, this is key).
    • If your protein of interest has a high molecular weight, a lower percentage gel is the best choice.
    • If your protein of interest has a low molecular weight, a higher percentage gel is the best choice.
      • Note: You can also look online to see how proteins of different molecular weights will separate depending on the type of gel and running buffer used.
      • Precast gel note: Remove any stickers that say, “Remove before use.”
  • Prepare running buffer.
    • Note: It’s best to make and use fresh if you can—there are so many places for error in Western blotting and starting off with running buffer that’s stinky or depleted of something important would be super sad!
  • Fill your tank.
    • I know, you can run your gel faster with less buffer—but you can also melt it. (Smiling Western blot bands, anyone?)
  • Load your ladder and samples.
    • Vortex, vortex, vortex—unless you want your protein concentrations to be off because your samples settled.
    • If you have empty wells in your gel, fill them with some loading dye—this will help the gel run straight and will also counteract those smiling bands.
    • Note: If you’re going to cut your resulting membranes vertically to test more than one set of samples, standards and ladders are great ways to separate the sets and to maintain the correct orientation of your samples.
  • Run the gel.
    • Every system is different—consult the interwebs for the optimal voltage, time, and temperature to run your gel based on the buffers and equipment being used. (For example, should you run it overnight in the cold room at low voltage or at room temperature for a couple of hours?)
      • Ahhh, we’ve come to one of those steps: Do you want results ASAP? Do you need to do a ton of other stuff and would rather set it and forget it? You can take this into consideration, too.
    • If you consistently fill your tank to the “full” line, you can determine the exact voltage time that it will take to run your dye front right to the bottom of the gel every time. This ensures good separation of your proteins, that neither your samples or your molecular weight standards have disappeared into the abyss and that you can easily pass off your protocol to others: “Just run it at ______ for _______.”
  • Prepare for transfer.
    • All membranes are not created equal! Choose the appropriate membrane for your experiment—PVDF or nitrocellulose? What size pore? Phone a friend, ask another lab member or consult the internet for the best option based on your protein of interest—especially if it’s gargantuan or teeny tiny (or if your lab has a bunch of different membrane types laying around).
    • Activate your membranes if needed—it takes seconds, but is super important.
    • Tip: I cut the top left corner of each membrane on a diagonal to always keep orientation.
    • Note: Never touch the membrane with your fingers—even if you’re wearing gloves! Find, steal, order, (whatever) yourself a nice pair of flat headed tweezers to use for this, instead.
  • Build your “transfer sandwich.”
    • Remove your gel cassette from the tank and separate the cassette to expose the gel.
      • Tip: Tanks have a weird shape to their lids and gels have a slot across them so that you can easily break apart the plastic without ripping your gel.
    • Build your “transfer sandwich.” Five years in I am confident that I alwaysPicture1 build my sandwich in the correct order, but this was not always the case. Aftera few “Where the hell did my proteins go!” moments, I devised my little schematic. This is for our dry transfer apparatus, but you get the picture. It also alleviates a ton of questions from new users second guessing their build.
      • Be gentle but firm when creating your masterpiece—then pray for no bubbles!
  • Choose transfer times and voltages.
    • You should transfer low molecular weight proteins for less time or at a lower voltage so your proteins don’t go through the membrane.
    • Higher molecular weight proteins can transfer for longer time or at a higher voltage to give them time to migrate into the membrane.
    • Voltage time for transfers will vary based on your choice of buffers, membranes, equipment (wet versus dry transfer) and temperature.
      • Again, the webpage of your favorite Western blot company is helpful here.
      • And yet another place for deciding on whether to continue moving forward or take a pause by transferring overnight…
  • Block your membrane.
    • It’s best to check with the company that developed your antibody for the optimal conditions, but here are some blocks I’ve encountered:
      • 5 minutes in methanol at room temperature followed by allowing the membrane to dry completely (meaning you can dry overnight if you need to head out for the day).
      • 1 hour in 5% nonfat dry milk (NFDM) or 5% BSA in PBS or TBS.
      • 1 hour in membrane blocking solution (usually a company’s code name for some type of serum).
  • Prepare your antibodies.
    • I’ve always been taught to prepare your antibodies in whatever solution you blocked in. However, this can vary based on your blocking solution—for example, you may be better off diluting your antibody in 2% instead of 5% NFDM.
    • Primary antibody:
      • Consult the developer’s website for a dilution range in which to start. Around 1:1,000 is a good bet if you don’t have a solid starting point—unless you’ve got a low-abundance target.
      • Incubate overnight on a shaker in a cold room, or incubate for 1-4 hours on a shaker at room temperature.
        • Alas, another potential stopping point.
    • Secondary antibody:
      • A 1:5,000 or 1:10,000 dilution seems is standard for HRP-linked secondaries. This will differ if you are using fluorescently-conjugated secondaries—ask the Google!
  • Cross your fingers and toes, and think positive thoughts!

While Western blots are a super common lab technique, there are many places where they can go awry. Working out a standard protocol for the proteins of interest and antibodies most often used in your lab can eliminate a lot of the hubbub; so too can a trusted troubleshooting guide for when those blots are dirty, blank, running crooked, or smiling. Cheers!


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Amy M Palubinsky

Amy M Palubinsky

Amy Palubinsky is a Neuroscience PhD candidate at Vanderbilt University and mom to two human children and one fur-kid. She hates mornings and therefore loves coffee.