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Western Blotting Protocol

Western-blot protocol

Preparation of Samples for SDS-PAGE Gel Electrophoresis from Prepared Cell Lysates

  1. Prepare gels for SDS-PAGE
  2. Prepare 1X running buffer
  3. Add 50 mL of beta-mercaptoethanol to 950 ml sample buffer (for 1 mL). (only if you have not pre-added beta-mercaptoethanol to sample buffer)
  4. Add sample buffer to each sample (pre-determine how much sample you can load per well - BioRad thin combs can retain about 30uL. Large combs can accomate up to 50uL).
  5. Vortex samples briefly.
  6. Poke a hole in cap of each tube (to prevent the tops popping when boiling)
  7. Boil in heating block/water bath at (95°C) for 5 minutes (lower temperatures have been shown to prevent non-specific protein aggregation)

After Boiling Protein Samples - Loading and Running the SDS-PAGE Gel

  1. Centrifuge samples for 1 minute at high speed.
  2. Load sample into each lane.
  3. Load MW (molecular weight ladder) reference. (you may want
  4. Run gel at 100V (100V through stacking gel, voltage can be increased up to 200V when running in separating gel with plenty of running buffer)

Transfer of Protein Samples to Membrane

  1. Prepare 1X Transfer buffer
  2. Soak and shake gel in Transfer buffer for 15 min
  3. Soak nitrocellulose membrane (or PVDF) and filter paper (extra thick) for several minutes.
  4. Place sandwich on transfer cell:

Extra thick filter paper CLEAR
Membrane
Gel
Extra thick filter paper BLACK

  1. Electric transfer: 35V overnite or 90V for 1 hour depending on the size of your protein or your patience!

Western Blotting Protocol or Immunoblotting

  1. Prepare 1X TBST from stock solutions.
  2. Prepare blocking buffer (3% Bovine Serum Albumin or 5% Blotting-grade milk (Blotto) in TBST). (you may want to try several different blocking times and conditions).
  3. Wash membrane in 1X TBST with shaking for 10 min.
  4. Block at room temperature for 1 hour with blocking buffer (shaking or circular rotator)
  5. Incubate your membrane (PVDF or nitrocellulose) with primary 1° Ab antibody overnight (for difficult blotting conditions ie phosphorylation of proteins) or 1 hour for ( easy conditions such as total protein) at 4°C (overnite) or room temperature (1 hour incubation only) covered with saran wrap (gentle shaking). You can use plastic tubs from the dollar store for this (use these only for blotting!) or use pipette box bottoms. The dilution for primary antibody is around 1:1000. See manufacturer's instructions.
  6. Wash with 1X TBST  3 X 15 min
  7. Incubate your membrane (PVDF or nitrocellulose) with secondary antibody, 2° Ab for 30 – 45 min or (1 hour - for difficult blotting conditions) at room temperature. The dilution for secondary antibodies is usually 1:10,000 or higher. (ie 1uL of secondary in 10mL of TBST per membrane) See manufacturer's instructions.  You may want to add your antibody to blocking solution to decrease non-specific binding especially if you got high background in your first experiment.
  8. Wash with TBST   4-5 X 10 min. This is the most important washing step.
  9. ECL (5min)
  10. Expose to film. Usually exposure time of western blots is about 10-30 seconds. Longer (5-10 minutes) for phosphorylation. If you have a really weak signal, either you need to load more protein or try to increase exposure time (up to 30 minutes - you can try leaving it in a casette longer).
  11. Keep your membrane! You can keep your membrane in TBST 1X in the fridge for a while. You may need it for the following reasons: 1) checking loading (paper reviewers may ask for total protein or a loading standard such as actin). Also, you can probe initially for a phospho-protein and then strip and reprobe for total protein.

Also see our Western Blotting Membrane Stripping Protocol

Our Protein Protocols


 

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