Category: Protocol
Spandau Lab
IMMUNOBLOTING
SDS-PAGE Separating Gel
Part A
(1a)
Assays are normally performed on keratinocyte cultures that are growing in 100 mm dishes. At the
desired time point, the dishes are removed from the incubator and treated as follows;
(2a)
Wash plates 2 x with 5ml of ice-cold PBS.
(3a)
To harvest cells several methods maybe used:
(4a)
i)
Remove PBS and add 1.0 ml of RIPA Buffer containing Complete Mini Protease Inhibitor
Cocktail (Roche # 1836153), sodium orthovanadate and sodium fluoride. Incubate plates on ice
for 15 minutes with occasional swirling. Scrape cells from the plate and transfer to an Epindorf
tube, sonicate cells 10-bursts on ice. Centrifuge full speed, transfer supernatant to new tube and
determine protein concentration (BioRad Bradford method). Add Laemmli Sample Buffer
(ratio1:4).
ii)
For 100mm dish of T75 flask lyse cells directly using 500ul of ice cold Laemmli Sample Buffer
containing b-mercaptoethanol (BioRad #161-0737), Complete Mini Protease Inhibitor Cocktail
(Roche # 1836153), sodium orthovanadate and sodium fluoride. Scrape cells from surface and
transfer to an epindorf tubes on ice. Sonicate 10-bursts on ice, centrifuge 5min full speed and
determine protein concentration using detergent compatible BioRad kit (Lowry method).
Determine the protein concentration of samples
Bradford method: (BioRad # 500-0006) at 595nm
Lowry method: (BioRad kit # 500-0119) at 730nm detergent compatible
Bradford method 595nm:
Standard Curve = 750ul dH2O + 5, 10, 15, 20, 25 or 30ul of BSA (0.4ug/ml) Sigma P7656 + 200ul BioRad dye
Samples = 750ul of dH2O + < 3ul sample + 200ul BioRad Protein dye
Page 1
Category: Protocol
Spandau Lab
Part B
(1b)
Prepare an SDS-PAGE separating gel (for BioRad Mini Protean II gels – 6ml enough for 1-gel)
CONCENTRATION
40% Acrylamide/Bis (BioRad)
Separating Buffer (BioRad)
Distilled Water
Stacking Buffer (BioRad)
10% Ammonia Persulphate
TEMED
TOTAL
7.5%
SEPARATING GEL
10%
12%
STACKING GEL
4.0%
2.25 ml
3.0 ml
6.75 ml
-
3.0 ml
3.0 ml
6.0 ml
-
3.6 ml
3.0 ml
5.4 ml
-
0.6 ml
3.9 ml
1.5 ml
60 ul
30 ul
6 ml
60 ul
30 ul
6 ml
60 ul
30 ul
6 ml
60 ul
30 ul
6 ml
(2b)
Put on samples on heat block 100oC for 5m min,
Meanwhile thaw Kaleidoscope marker (BioRad #161-0324)
(3b)
Load sample on SDS-PAGE gel (see above) and run 150volts, approx. 1hr 30min
Page 2
Category: Protocol
Spandau Lab
Reagents Required
RIPA buffer with Pefabloc SC and NaOrthovanadate
For 100 mls
150 mM NaCl
50 mM Tris, pH 8.0
1 % NP-40
0.1 % SDS
0.5 % NaDeoxycholate
3 mls of a 5 M stock
5 mls of a 1 M stock
10 mls of a 10% stock
0.1 g SDS
0.5 g Sodium Deoxycholate
!! Autoclave RIPA buffer !!
Before use add either:
(a) Pefabloc (1 ul per 1 ml RIPA); “activated” NaOrthovanadate (37 ul per 1 ml RIPA); Sodium Fluoride
(5ul per 1ml RIPA) to the required amount of buffer.
– OR –
(b) Complete Mini Protease Cocktail Inhibitor (Roche #1836153 - 1 tablet in 7ml = 1x); “activated”
NaOrthovanadate (37 ul per 1 ml RIPA); Sodium Fluoride (5ul per 1ml RIPA) to the required amount of
buffer.
TBS (pH 7.5)
TBS Tween-20 (pH 7.5)
150 mM NaCl (15 ml of 5 M/500 ml)
100 mM Tris, pH 7.5 (25 ml of 2 M/500 ml)
**or buy 10X TBS (BioRad 170-6435)
150 mM NaCl (15 ml of 5 M/500 ml)
100 mM Tris, pH 7.5 (25 ml of 2 M/500 ml)
0.2% Tween-20 (1ml Tween-20/500ml
**or buy 10X TSB (BioRad 170-6435)
Western Stripping Buffer
Blocking solution (100ml)
62.5 mM Tris, pH 6.8 (12.5 ml of 0.5 M/100 ml)
2 % SDS (20 ml of 10%/100 ml)
100 mM β-mercaptoethanol (710 ul/100 ml)
5g non-fat dry milk (Carnation Brand)
100ml TBS
Running Buffer (5x stock)
Transfer Buffer
45.0grams
Tris-Base
216grams
Glycine
15grams
SDS
3 liters
ddH2O
For use dilute 300ml 5x stock with 1.2L ddH2O
** or buy 10X from BioRad #161-0772
** buy 10X from BioRad #161-0771
Laemmli Sample Buffer (8ml)
3.8ml
ddH2O
1.0ml
0.5M tris-HCL, pH 6.8
0.8ml
Glycerol
1.6ml
10% SDS
0.4ml
B-mercaptoethanol
0.4ml
0.05% (w/v) bromophemol blue (in water)
**** or purchase from BioRad #161-0737
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