Biotechnology and Bioinformatics (1+1)

Materials

i) Western Blot apparatus and accessories

ii) SDS - PA G E

Acrylami de stock (30%)

1 .5 M Tris- H CI (pH 8.8)

1 M Tris-HCl (pH 6.8)

10% Ammonium Persulphate 10% SDS

TEMED (N , N , N', N' – Tetramethyl ethylenediamine)

Tris, Glycine , SDS-Running buffer

Sample Buffer 4X

Water saturated n-butanol

Note: Polyacrylamide gels are formed from the polymerization of two compounds, acr y lamide and N, N-methylene bisacryl a mide (Bis, for short). Bis is a cross-linking agent for the gels. The polymerization is initiated by the addition of ammonium per sulfate along with either DMAP or TEMED. The separation of molecules within a gel is determined by the relative size of the pores formed within the gel . The pore size of a gel is determined by two factors: the total amount of acrylamide present (designated as % T) and the amount of cross-linker (%C) . As the total amount of acrylamide increases, the pore size decreases.

Rule of thumb: The smaller the size of the protein of interest, the higher the percentage of polyacrylamide (mono:bisacrylamide). The bigger the size of the protein of interest , the lower the percentage of mono: bis .

iii)Western blotting

PBS (10X)

Transfer Buffer

Blocking solution

Primary antibody

Secondary antibody

Developing solution

II. Preparation of stocks

i) A c ryla mide 30% ( 1 0 0 ml)

A c ry lamide : 2 9 . 2g

N -N bis acrylamid e : 0. 8 g

The above mentioned sub stances are dissolved in 100ml steriled is tilled water and stored in brown bottle at 4 ° C .

ii) 1.5M Tris-HCI

18.171 g Tris is dissolved in 100 ml water. The pH is adjusted to 8 .8 using HCl until it stabilizes. Then it is filtered through Whatman No . 1 filter paper and auto claved. (Not e : Tris-HCl can be stored at room temperature )

iii) 1M Tris- HCI

6.057g Tris is dissolved in 50 ml water . The pH is adjusted to 6 . 8 using HCI until it

stabilizes and then filtered through Whatman No. 1 filter paper and autoclaved . (Note: Tris-HCl

can be stored at room temperature)

iv) 10% SDS

10 SDS is dissolved in 100 ml of distilled water .

v) SDS gel running buffer

Tris : 3 g

Gl y cine : 14 . 4 g

S DS : 1 g

vi) Sample loading buffer 4X for 5ml

1M Tris HCL (pH 6.8) : 2.1ml

100% Gl yc e r ol : 1.0 ml

β-Mer c a p t o ethanol : 0.5ml

Bromophenol blue (0.05%): 2.5mg

The above mentioned substances are dissolved and made up to 5 ml with steriled is tilled water.

vii) Water saturated butanol

Equal volumes of n-but a nol is added to distilled water and mixed well and allowed it to stand for 10 minutes.

viii) l0%APS

0 . 1 g of ammonium persulfate is dissolved in1 ml of water .

Precaution: APS to be freshly prepared before every use

	ix) 5% Stacking gel (2ml)
	30% Acrylamide	0.33 ml
	1 . 0 M Tris (pH ~ 6.8)	0.25 ml
	10% SDS	0.02 ml
	1O%APS	0.02 ml
	TEMED	0.002 ml

The above mentioned substances are dissolved and made, up to 2ml with sterile distilled water.

x) 10% Separating gel (5ml)

Acrylamide 1.7 ml

1.5 MTris (pH - 8 . 8) 1 . 3 ml

SDS 0 . 05 ml

APS 0.05 ml

TEMED 0.002 ml

The above mentioned substances are dissolved and made upto 5ml with sterie distilled water .

xi) l0 X PBS (1 L)

NaCl : 80g

KCI : 2g

Na₂H P0 _{4 :} 11.5g

KH₂P04 : 2 g

Distilled water : 1000ml

xii) Transfer Buffer ( 1 L)

25m M T ris :2 .9 g

190m M G l ycin e : 1 4.5g

20% MethanoI : 200m l

Dissolved in IX PBS. : 800ml

xiii) Blocking Solution

5% non-fat dry milk and 0 . 1 % Tween 20 are dissolved in Ix PBS

xiv) Staining solution

Coomassie Brilliant Blue (R-250) : 0.001 %

Methanol : 50 %

Acetic acid : 7%

xv) Destaining Solution

Methanol : 50 %

Acetic acid : 7%