Western Blot Extras

Alkaline Phosphatase (AP) Developing

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

Incubate in stripping buffer for 30 minutes at 50-70°C.
Wash membrane 2X in TBS-T.
Incubate with SuperSignal® to verify it has been stripped completely.
Wash 2X for 10 minutes in TBS-T.
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.

Incubate in mild stripping buffer for 1 hour at 37°C.
Wash membrane 2X in TBS-T.
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.
Wash 2X for 10 minutes in TBS-T.
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

Take gel and place in Coommassie for 2 hours to O/N.
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.
To prevent cracking, you can add 1% glycerol to the last hour prior to drying the blot.