Western Blotting
SDS-PAGE Protocol with BioRad Gel Assembly
Gel protocol
Prepare 1X Running Buffer (RB) from 5X solution (100 ml/run)
Assemble gel apparatus with BioRad pre-cast gel (see manufacturer’s protocol)
Cut sealing strip from bottom of pre-cast gels
If using only one gel, use blank assembly for other side
Electrophoresis Tank
Running Buffer
Gel Holding Tank
Black Contact
Blank, If Only
One Gel
Red Contact
Gel with
Wells to
Inside
Locking Tabs
Pour 1 L of 1X RB into tank
Remove comb from gel (carefully)
Wash wells with RB
Make sure no air bubbles in wells or below gel
Use 5% or 10% Tris-Cl gels
Combine 50 ug (or maximum possible) solubilized sample with 5X SDS-Loading Buffer
Heat to 85° C for 5 minutes
Well capacity is ~ 50 uL
Load 10 uL Kaleidoscope Standard Proteins + 2 uL 5X SDS-Loading Buffer
Eluted samples don not have to be heated to 85 C again
Optional:
Add 10 uL diluted, heat denatured Biotinylated Standards to well, with 5X SDS-Loading
Buffer. May contribute to background
Run at 150 V for 45 minutes at RT
Make sure wells remain submerged below the RB
Do not run standard beyond gel bottom
Prepare 1X Transfer Buffer (TB)
Use cold qH2O
Methanol shrinks the membrane pore size
Supplement TB with 1% Tween-20
Facilitates transfer of proteins larger than 100kDa
Prepare PVDF membranes, cut to size of gel (simultaneously with next step)
Pre-wet membranes in 100% methanol, then incubate in TB for 10 minutes
Trim one corner to mark protein side
Use TBST for PVDF membranes (smaller proteins)
Trim gel and prepare “sandwich”
Cut 0.5 cm off bottom and all wells off top
Soak gel, blotting papers and filter pads in TB for 10 minutes
Assemble sandwich in TB, rolling and wetting each set
Do not let membranes go dry
Assemble as shown below:
White Side
Fiber Pad
Blotting Paper
Membrane
Gel
Blotting Paper
Fiber Pad
Black Side
Place “sandwich” in tank, along with ice block
Transfer Tank
Transfer Buffer
Ice Block
Red Contact
Stirrer
Black Side
White Side
Blotting
Apparatus
Holder
Black Contact
Blotting “Sandwich”
Apparatus
Locking
Tab
Transfer at 40 V overnight or 60 V for 3 hours
Perform in cold room, while stirring constantly
Protein standards should be completely transferred from gel to protein when done
Rinse membrane with TBST
Block non-specific interactions with 5% milk in TBST
Supplement with protease inhibitors
Incubate overnight at 4° C or 1 hour at room temperature
Place membrane in pipette tip box lid. Cover with aluminum foil and label
Milk blocking solution should be freshly prepared
Tween-20 helps reduce background
Membrane is viable for 1 week if stored in 1X TBST at 4° C
Continue as follows…
Blotting Protocol
Wash membrane 2X quick, 3X-15 minutes, 2X-5 minutes; all with shaking in TBST
Use largest volume of wash buffer possible
Add primary antibody at appropriate dilution, incubating 2-3 hours with shaking
Anti-GFP Ab – diluted 1:1000 in 1X TBST
Do not let membrane go dry after incubation
Save primary antibody solution for re-use
Wash membrane 2X quick, 3X-15 minutes, 2X-5 minutes; all with shaking in TBST
Add secondary antibody, 1:2000 for 1 hour
7.5 uL secondary Ab/15 ml 1X TBST
If RIM2 Ab, dilute 1:500 in 1X TBST
To save on antibody, use smaller volume
Place membrane in plastic weighing boat, add just enough volume to cover
Wash membrane 2X quick, 3X-15 minutes, 2X-5 minutes; all with shaking in TBST
Add tertiary antibody, 1:2500 for 1 hour
6 uL tertiary Ab/15 ml 1X TBST
Wash membrane 2X quick, 3X-15 minutes, 2X-5 minutes; all with shaking in TBST
ECL Detection
Wear powder free gloves
Place blot flat on Saran wrap with protein side up
Blot corner of membrane to remove excess buffer
Pipet ECL reagents (1 Luminol:1 H2O2 (v/v) onto protein side of membrane
Use just enough reagent to cover membrane
Incubate exactly 1 minute, without agitation
Work quickly from this pont
Drain membrane, blotting corner to remove excess reagent
Cover membrane completely with Saran wrap
Trim excess
Smooth away air pockets
Develop image (film, etc.) in darkroom
Apply protein side of membrane against ECL film
Incubate as needed
Stripping Membranes
Incubate membrane in Strip Buffer (SB) for 30 minutes at 70° C
Wash 1X with TBST
Re-blot for second protein as in steps 12-20 above
Reagents
5X Running Buffer (RB)
15 g
Tris Base
72g
Glycine
5g
SDS
To 1 Liter
H2O
Do not pH (8-9)
Stripping Buffer (SB)
750 uL
B-mercaptoethanol
2g
SDS
To 100 ml
1X TBS
10X Transfer Buffer (TB)
58 g
Tris Base
29 g
Glycine
To 1 Liter
H2O
10X TBS
200 ml
Tris Base
80 g
NaCl
To 1 Liter
H2O
1X TBST
1 ml
Tween-20
999 ml
1X TBS
To 1 Liter
H2O
Do not pH (8-9)
pH to 7.6
Mix Well
Trituration Buffer
2%
Triton X-100
10 mM
Tris Base
1 mM
EDTA
Protease inhibitors
Mix Well
Lysis Buffer
0.5 %
SDS
0.05M
Tris-Cl
1 mM
DTT
Protease inhibitors
pH to 8.0
TBST+ 5% Milk
2.5 g
Dry Milk
To 50 ml
1X TBST
Protease Inhibitors
Name
PMSF(Phenylmethanesulfonyl Fluoride)
Aprotonin
Leupeptin
Pepstatin
Benzmidine
Pefabloc SC
Calpain Inhib. I+II
[Working]
1.5 mM
10 µg/ml
10 µg/ml
10 µg/ml
1 mM
0.4 mM
8 µg/ml
V for 10ml Buffer
30 µL
6.7 µL
20 µL
10 µL
10 µL
10 µL
8 µL
[Stock]
0.5 M
1.5 mg/ml
5 mg/ml
10 mg/ml
1M
400 mM
10 µg/µL
Stock Storage
-20° C
4° C
-20° C
-20° C
-20° C
-20° C
-20° C
Storage Media
H20
H20
H20
H20
H20
H20
(EtOH)