Protocols

Detection of IkB by Western Blotting

Posted on Saturday, October 18, 2003

Description
IkB was detected by western blotting, the cells were washed, lysed by RIPA buffer, protein extimated by pattersons method and run on an SDS-Page (polyacrylamide gel electrophoresis) gel, blotted onto nitrocellulose membrane and detected by ECL reagents.

Procedure
The cells (5 x 106) were washed twice in ice cold PBS and suspended in RIPA buffer for 30 minutes, centrifuged at 12,000 rpm. The supernatant was transferred to another tube. The protein estimation was performed.The lysate (1 in 100 dilution) along with standard solutions of BSA were taken (10-100 mg/ml) at a final volume of 100 ml in a microtitre plate, working solution A was added (100ml) and the plate incubated at room temperature for 10 minutes. This was followed by addition of 50 ml of working solution B and a further incubation of 30 minutes, following which plate was read on an ELISA Reader at 690 nm and a standard plot was drawn. Lysate (100mg) was loaded on to a 15% SDS PAGE, run and blotted onto a nitrocellulose paper(Hynond ECL, APB, USA). The blot was blocked in blocking reagent, incubated overnight in primary antibody (1 in 300 dilution), washed twice in TBS with 0.05% Tween-20, incubated with secondary antibodyfor one hour, washed 4 times and developed using ECL Reagents as per manufacturers instructions.

Recipes
1. PBS: to be prepared as described previously.
2. RIPA Buffer: was prepared freshly before use. For 10ml
10 mM Tris (pH 8.0), (1ml of 100mM Tris pH 8.0)
0.5% NP-40, (50ml)
250 mM NaCl, (0.5ml of 5M NaCl stock)
10 mM Sodium Deoxycholate ( gms) [all from (SRL, India)]
3. For Protein Estimation
a. Working Solution {A}
(a) Copper-tartarate-carbonate solution
1. 20% Sodium carbonate:- 20gms in 50 ml D/W à100ml
2. 0.2% CuSO4:- 0.2 gms CuSO4 in 40 ml D/W
3. 0.4% K-Tartarate:- 0.4 gms in 40 ml D/W
Mix solutions (2) and (3)à make volume upto 100 ml with D/W
Then add solution (1)
(b) 10% SDS solution
(c) 0.8 N NaOH solution (all from SRL, India)
To make Working Solution {A}
Add (a) + (b) + (c) + D/W : : 1 + 1 + 1 + 1
b. Working Solution {B}
Dilute 2N solution of Folin ciocalteau's Phenol reagent (Sigma, USA) 1:5 prior to use.
c. BSA 1mg/ml. (Sigma, USA)
4. For SDS Page
a. 15% Runing Gel 4 %Stacking Gel
30% Acrylamide (Sigma, USA) 7.5 ml 2.0 ml
1M Tris (pH 8.8) (SRL, India) 5.6 ml 1.9 ml (1Mtris pH 6.8)
D/W 1.75 ml 11 ml
10% SDS(SRL, India) 0.15 ml 0.15 ml
TEMED (Sigma, USA) 0.015 ml 0.015 ml
APS (10 mg/ml) (Sigma, USA) 0.1 ml 0.1 ml
TOTAL 15 ml 15 ml
b. Sample Buffer (10 ml)(Redu)
10%SDS(SRL, India) 4.0 ml
Glycerol(SRL, India) 2.0 ml
1M Tris (pH6.8) (SRL, India) 1.25 ml
D/W 1.75 ml
2-ME (Sigma, USA) 1.0 ml
c. Tank Buffer
For 1L
Tris Base (SRL, India) 3 gms
Glycine (SRL, India) 14.4 gms
10% SDS (SRL, India) 10 ml
5.For Western Blotting
a. Transfer Buffer (pH 8.3) 5L
25 mM Tris (SRL, India) 15.1 gms
192 mM Glycine (SRL, India) 72 gms
D/W to 4 L
20 % Methanol(SRL, India) 1 L
b. TBS (Tris Buffered Saline)
c. Blocking Reagent: 5% Milk Powder (Aniksprey) in TBS with 0.05% Tween 20 (Sigma, USA)
d. ECL Western Blotting Kit (APB, USA) used as per manufacturers instruction.
6. Antibodies: Primary antibody against IkB raised in rabbit was from Santa Cruz Biotechnollogy, USA and secondary antibody was horse ant- rabbit, peroxidase labelled


Supplies


Tips


Submitted by: manny440