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Western Blot Extras

Alkaline Phosphatase (AP) Developing

  1. Use secondary antibody conjugated to AP and incubate 2 hours.
  2. Wash 6X for 5 minutes in Tris buffered saline-Tween-20 (TBS-T).
  3. Put in 10 ml AP buffer + 66 µl NBT and 66 µl BCIP and develop.
  4. Place in stop buffer (PBS + 20mM EDTA) for 10 minutes to stop reaction.
  5. Lay on glass and dry blot in dark place.

AP Buffer

  • 100mM Tris, pH 9.5
  • 100mM NaCl
  • 5mM MgCl2

NBT

  • 1 g/20 ml 70% DMF
  • Store at -20°C

BCIP

  • 0.5 g Disodium Salt/20 ml
  • Store at -20°C

Stripping Blot for Reprobing: Stringent

  1. Incubate in stripping buffer for 30 minutes at 50-70°C.
  2. Wash membrane 2X in TBS-T.
  3. Incubate with SuperSignal® to verify it has been stripped completely.
  4. Wash 2X for 10 minutes in TBS-T.
  5. Block for 2-4 hours, then place into primary antibody O/N.

Stripping Buffer

  • 2% SDS
  • 62.5M Tris, pH 6.8
  • 100mM β-mercaptoethanol

50 ml

  • 10 ml 10% SDS
  • 3.1ml 1M Tris, pH 6.8
  • 347 µl 14.4M β-mercaptoethanol

Stripping Blot for Reprobing: Mild

Note: This has been used on 22% histone gels and reprobed 6-8 times before any loss of signal was noted.

  1. Incubate in mild stripping buffer for 1 hour at 37°C.
  2. Wash membrane 2X in TBS-T.
  3. Incubate with SuperSignal to verify it has been stripped completely. Expose to film for at least 1 hour, longer if it took longer to get the original signal.
  4. Wash 2X for 10 minutes in TBS-T.
  5. Block for 2-4 hours, then place into primary antibody O/N.

Mild Stripping Buffer

  • 2% SDS
  • 100mM Tris, pH 7.5
  • 14 mM β-mercaptoethanol

Staining Gels/Blots

Note: Stain blot with 1-2X Ponceau S, and destain with water. The stain will be very light, but you can then wrap the blot in Saran Wrap® and scan the stained blot into photo-editing software on the computer. Use the contrast function to darken the image to see the stain.

Coommassie Staining Gel

  1. Take gel and place in Coommassie for 2 hours to O/N.
  2. Destain gel until bands are clearly apparent. Add Kimwipes to destain to help absorb the dye without altering the make-up of the destain. Change Kimwipes® as they become full of dye.
  3. To prevent cracking, you can add 1% glycerol to the last hour prior to drying the blot.

Coommassie Dye (2.5 L)

  • 6.25 g Coommassie® Brilliant Blue G-250
  • 1.12 L Methanol
  • 250 ml Glacial Acetic Acid
  • 1.12 L H20
  • Store at room temperature

Destain (10 L)

  • 300 ml Glycerol
  • 2.5 L Methanol
  • 1 L Glacial Acetic Acid
  • 6.2 L H20
  • Store at room temperature

© 2014 The University of Texas MD Anderson Cancer Center