western blot protocols, troubleshooting, scientific resources and research atricles for the research community and science students


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Western Blot Buffers    
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 		   10 x SDS PAGE Buffer
a. Tris Base 60.55 g
b. Glycine 288.27 g
c. SDS 20 g
d. dH2O – complete up to 2 liters		  

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4. Western Blot Procedure Western blot protocol overview.

 

 Lysis Buffer
a. 5 mM Tris-HCl, pH 7.4
b. 2mM EDTA
c. 10 µl Protease Inhibitor Cocktail (1000 x stock)
i. 10 mg/ml benzamidine
ii. 5 mg/ml leupeptin
iii. 5 mg/ml trypsin inhibitor
PVDF Transfer Buffer
a. Trisbase 12.11 g
b. Glycine 57.65 g
c. Methanol – 100 ml
d. dH2O – complete up to 4 liters
Resuspension Buffer
a. 75 mM Tris, pH 7.4
b. 12.5 mM MgCl2
c. 5 mM EDTA
RIPA Buffer
a. 1% Igepal CA-630 (0.5 ml)
b. 0.5% Sodium deoxycholate (0.25g)
c. 0.1% SDS (0.05g)
d. PBS (49.5 ml)
Just prior to use add:
i. 10 mM beta-mercaptoethanol
ii. 10 ug/ml PMSF
iii. 5 ug/ml aprotinin
iv. 0.1 mg/ml benzamidine
v. 1 ug/ml pepstatin A
vi. 1 ug/ml leupeptin
vii. 100 mM sodium orthovanadate
viii. 15 ul/ml Triton X-100
TBS Buffer
a. Tris-HCl 12.11 g
b. NaCl 116.8 g
c. dH2O – complete up to 4 liters
d. pH 7.5 with Tris Base
TE Buffer
a. EDTA 0.2922 g
b. Tris Base 1.211 g
c. dH2O 1 liter
d. pH 7.4 with HCl
    Tris-EDTA-Nacl buffer
(25 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, pH 7.6)
a. 3.03 g Tris,
b. 0.29 g EDTA,
c. 8.77 g NaCl
d. dissolve in dd.H2O.
e. Titrate the solution to pH 7.6 using HCL,
f. Complete volume up to 1l with dd. H2O.
    3% Blocking solution
a. dissolve 3 g BSA in 100 ml Tris-EDTA-Nacl buffer
    Washing Buffer
(0.1% Tween20 in Tris-EDTA-Nacl buffer )
a. Dissolve 0.1ml Tween20 with 100 ml Tris-EDTA-Nacl buffer.
    Alkaline phosphatase detection buffer
(50 mM Tris-HCl, 5 mM MgCl2 ; pH 9.5).
a. 0.61 g Tris
b. 0.1 g MgCl2
c. dissolve in water
d. titrate pH and make up to 100 ml.
    Alkaline phosphatase substrate solution
(0.015% (w/v) BCIP, 0.03% (w/v) NBT in detection buffer).
a. Mix BCIP (1.5 mg in 1 ml DMF), NBT (3 mg in 1 ml alkaline phosphatase
b. detection buffer) and 10 ml detection buffer just before use.
c. store BCIP (5-Bromo-4-Chloro-3-Indolyl Phosphate) at -20°C and NBT (P-Nitro Blue Tetrazolium chloride) at 4°C.
       
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