Odom:Western Blot

Overview
This is the western blot protocol used in the Odom Lab. Standard protocol for use with pre-cast gels.

Sample Buffer

 * 950ul Laemmli Sample Buffer (Biorad)
 * 50ul BME

Running Buffer

 * 150ml 10X Tris/Glycine/SDS
 * 1350ml water

Transfer Buffer (10X mix, 20% methanol)

 * 200ml 10X Tris/Glycine
 * 400ml methanol
 * 1400ml water

Blocking Buffer (5% milk in TBS)

 * 2.5g nonfat dry milk (5%)
 * up to 50ml TBS

Ponceau S Solution

 * Dissolve 0.5g Ponceau S in 1ml glacial acetic acid
 * Bring to 100ml with water

TBST

 * 500ul Tween-20
 * 1L TBS

Primary Wash (0.5% milk, 0.01% NaAz in TBST)

 * 0.5g nonfat dry milk
 * up to 10ml in TBST
 * 20ul 5% sodium azide
 * primary antibody

Secondary Wash (5% milk in TBST)

 * 1g nonfat milk (5%)
 * up to 20ml with TBST
 * secondary antibodies

Procedure

 * 1) Add Sample Buffer to lysate and boil at 95 for 5min.
 * 2) * ~30-100ug protein/lane
 * 3) * 2 volumes sample buffer
 * 4) * PBS can be added to add volume to lysate
 * 5) Add Running Buffer and load gel
 * 6) Run at 60-100v for ~3 hours in the cold room
 * 7) Make transfer buffer and chill to 4
 * 8) Wet nitrocellulose membrane in ddH20 then transfer buffer for 1-15min
 * 9) Disassemble gel by pushing opening down onto lid of transfer apparatus
 * 10) Cut the stacking gel off
 * 11) Assemble transfer apparatus
 * 12) * Sponge
 * 13) * Whatman
 * 14) * Gel
 * 15) * Nitrocellulose membrane, pre-wet
 * 16) * Whatman
 * 17) * Sponge
 * 18) * Remove all air bubbles with each new layer
 * 19) * Membrane must be on positive side, gel on negative side
 * 20) Fill tank with Transfer buffer
 * 21) Transfer for 3.5 hours at 300mA or O/N at 100mA
 * 22) Disassemble apparatus and wet membrane in TBS for 5min
 * 23) OPTIONAL:  Visualize transferred proteins with Ponceau stain
 * 24) Place membrane in Ponceau S solution for 5min at RT
 * 25) Destain in water for 2min
 * 26) Completely destain in water for additional 10mins
 * 27) Block with blocking buffer for 2 hours at RT or O/N in cold
 * 28) Wash with TBST for 15min RT
 * 29) Block with Primary Wash for 3 hours at RT or O/N in cold
 * 30) Wash in TBST 4x15min at RT
 * 31) Wash with Secondary Wash for 1 hour at RT
 * 32) Wash in TBST 4x15min at RT
 * 33) Combine luminal solutions solutions 1:1 to make 5ml and drip over the membrane
 * 34) Cover with saran wrap and let sit in dark for 3min
 * 35) Dab off excess luminal and visualize in darkroom immediately
 * 36) * 2min is good starting exposure

Contact

 * cmc