for CAT isozymes  perform non-denaturizing gel electrophorse at 100 V for 2 hrs at 4 °C.  then  soak gel in 3.27 mM H2O2 for 15 min, rinsed with water, and stained in 2% potassium ferricyanide (10 min) followed by 2% ferric chloride (10 min) to visualize the bands.

for APX isozymes perform non-denaturizing gel electrophorse at 100 V for 2 hrs at 4 °C. then soak gel in 0.1 M sod. phosphate buffer(pH-6.2) containing 4 mM Ascorbic acid and 4 mM  H2O2 for 15 min, wash gel with d/w, then finally stain with solution of 0.125 M HCl containing 0.1% (w/v) potassium ferricyanide and 0.1% (w/v)ferric chloride .

i have used this protocol for salt stressed pearl millet. hope this will help you.  

thank you sneha...but 2 hours is enough for these CAT and APX isozymes?...in many protocol, i saw long duration nearly 12 to 22 hours in four degree... 

From my experience, it will depend of the size of the gel. If you use the mini-gels (10%; 100 to 120 V), probably 2 to 3 hours will be sufficient. It also depends on the complexity of your samples. But you can keep an eye on the gel and end the run at any time.

Dear Jey 

With my experience u can go through this procedure, it will help u lot, all my PAGE experiments i follow this.............

Polyacrylamide Gel Electrophoresis (Native PAGE).

PRINCIPLE:

Acrylamide is a monomer cross links with another monomer of acrylamide with the help of N, N’ methylene bis acrylamide, cross linker, to form polyacrylamide gels. This reaction is carried out or enhanced by the presence of ammonium per sulfate (APS) and tetraethylene methylene diamine (TEMED). In this APS acts as a catalyst and TEMED as initiator. The basic principle involved in the separation of the molecules in PAGE is called isotachophoresis (iso- same; tacho- speed; phoresis-separation). The PAGE is performed using discontinuous buffer system, moreover the large pore size in the stacker (3.5%) and 6.8 pH facilitates all protein molecules which are in distributed in the solution gets accumulated near the separating gel. As they start entering into the separating gel (10%) and pH 8.8 facilitates more and more glycinate ion formation. Since, the Cl- ion concentration is fixed, initially the Cl- ion has greater mobility than the glycinate ion and at place when chloride ion and glycinate ion concentration becomes equal both start moving at equal speed. Now then all the protein molecules which are having variable negative charge will separate according to their charge, molecular weight and shape. Thus, separated molecules are visualized by staining the gels with coomassiee brilliant blue R250 prepared in methanol: acetic acid: water (4:1:5 v/v/v). Here, the negatively charged proteins are precipitated by H+ released from acetic acid and methanol shirnks the pore size of the gel thus, precipitating the proteins. Coomassiee brilliant blue R250 binds with the peptide bond of the protein molecules.

Materials:

Lower Gel Buffer

1.5 M Tris-HCl; pH 8.8

Upper Gel Buffer

0.5 M Tris-HCl; pH 6.8

30% Acrylamide Stock.

29.2 g acrylamide

0.8 g N, N'-methylene-bis-acrylamide

dH2O to 100 ml volume.

Dissolve (as the acrylamide and bis-acrylamide takes up water therefore the salts are first dissolved in half of the original volume and later make up the volume to 100 ml) it, filter and remove particulate matter and store at 40C.

10% Ammonium persulfate solution.

Dissolve 0.1 g of ammonium persulfate in 1 ml distilled water.

Running Buffer

0.025 M Tris-HCl

0.192 M Glycine; pH 8.3

2 X Sample Buffer

20% Glycerol

0.125 M Tris-HCl; pH 6.8

0.1% Bromophenol blue

Method:

Prepare the reaction mixture in the following given composition. However, Add APS and TEMED at the last and gently mix the solution and pour them slowly in between the glass paltes. Now, apply 100 v/ 15 cm length gel.

 Run the gel at 50 milli Amps for 2 hrs or untill the marker dye reaches the end of the gel.