Western blot

Overview

• For the presence and relative abundance of protein.
• Also known as immunoblotting.
• Named as a play of words on Southern blotting (for DNA visualization) that was developed by Edwin Southern, thus spelled with a lowercase "w".
• Main steps:
  1. Transfer protein from a gel to a membrane by applying electric current.
     1. Proteins are coated with SDS and thus have a negative charge.
     2. The membrane goes under the gel, soaked filter paper is placed above and below, and the whole thing is sandwiched between two electrode plates with a positive electrode below.
  2. Block (passivate) membrane with BSA or skimmed milk powder to prevent non-specific antibody binding.
  3. Probe:
     1. Treat with primary antibody that specifically binds to your protein.
     2. Treat with a secondary antibody that binds to the primary antibody and allows to visualize it. Sometimes primary antibodies are already ready for imaging though.
  4. Image.
• Recommended to run SDS-PAGE with a prestained protein ladder so that you can see if the band you get is in the correct place.

Reagents

2X 1-step transfer buffer

• 39.02 g Tris-base
• 93.4 g glycine
• 56.16 g tricine
• 10 mL 0.5 M EDTA (pH=8)
• Milli-Q to 1 L

10X phosphate-buffered saline (PBS) buffer

• 80.06 g NaCl
• 2.01 g KCl
• 14.2 g Na2HPO4
• 2.45 g KH2PO4
• Milli-Q to 1 L

1X Wash buffer

• 998 mL 1X PBS
• 2 mL Tween-20

Blocking solution

• 10 mL 1X Wash buffer
• .4 g of skimmed milk powder

Procedure

1. Preparation:
   1. Cut out 4 Wattman paper pieces (10 x 8 cm).
      • Don't place the paper on the table, have the table lines with tissue paper.
   2. Cut 1 piece of PVDF membrane (9 x 7 cm).
      • Pore size should match the size of your protein. E.g., for 160 kDa SpCas9, the pores should be .45 um. But for small proteins (<15 kDa), use .2 um pore size.
      • Work with gloves on to prevent protein contamination.
   3. Add 20 mL of 2X Transfer Buffer and 20 mL Milli-Q into an empty gel box.
   4. Add 40 mL of methanol into another empty gel box.
      • Methanol can be reused, so don't throw it away at the end.
   5. Take out the SDS-PAGE gel, cut off the stacking part of it, and soak the resolving part in the 1X Transfer buffer.
      • This step replaces SDS running buffer with the transfer buffer.
2. Transfer (fast semi-dry method):
   1. Soak the Wattman paper in the 1X Transfer buffer and place two consecutive papers on top of each other, and on a special white plate.
   2. Soak the PVDF membrane in methanol.
      • Make sure it is submerged and when you take it out, do not let it dry!
   3. Place the PVDF membrane to the 1X Transfer buffer, make sure it is submerged (it is a bit hydrophobic so will need to rock it constantly), and place it on the Wattman papers.
      • There might be some bubbles on the membrane at this point..
   4. Place the SDS gel on the PVDF membrane and gently roll out any bubbles with a roller.
   5. Place two more Wattman papers soaked in the 1X Transfer buffer.
   6. Pour the leftover 1X Transfer buffer on the top of the last paper sheet.
   7. Put on the other (metal) plate, gently press down until you hear a click sound, and place it in the machine.
   8. Choose a present program or set your own.
      • For smaller proteins, 25 V / 1.3 A for 10 min is a typical setting.
      • For larger proteins, consider extending run time to 30 min for an increased protein transfer.
3. Blocking:
   1. Transfer the PVDF membrane to a clean box with a blocking solution in it and gently shake on a shaker for at least 30 min.
      • Longer blocking will reduce sensitivity while short blocking will increase background.
   2. Add antibody in the blocking solution with the membrane and leave on the shaker for at least 1 hour.
      • Choose the antibody based on your target protein. E.g., if your protein contains a StrepTag II, use 2.5 uL StrepTag II Antibody HRP Conjugate, which will result in a 4000X dilution.
      • If you know you will do this again, pour off the blocking solution with the antibody and freeze it in -20C for further use.
   3. Repeat 3 times: Pour 10 mL of the 1X Wash buffer on the membrane and gently rock for 10 minutes.
4. Imaging:
   1. Add 750 uL of chemiluminescence reagents (SuperSignal™ West Femto Maximum Sensitivity Substrate) from each of the two bottles (peroxide and luminol).
      • This step is performed in front of the imager – do not wait a long time after adding the reagents.
      • There is no need to rock it for long, just rock it in your hands for 10-15 seconds and then proceed with imaging.
      • HRP (horseradish peroxidase) uses H2O2 to oxidize luminol, which then emits blue light (425 nm).
   2. Have a document folder ready (cut it smaller and make it so it could open like a book so you could add the gel inside) and place the membrane inside of the folder before imaging.
   3. Image first with chemiluminescent setting on a dark plate for blotting, then image with the "fast blast" (white light) setting to get the ladder.
      • You may need to manually choose the exposure instead of the auto-exposure. Go for the highest or second-to-highest resolution.

Resources

• ThermoFisher Pierce Power Station manual