D.SABARIANNAI

1

• BY
• D.SABARIANNAI

2
AIM OF THE STUDY

• To screen the cyanobacteria which tolerate high
  salinity.
• To study what are the Antioxidant enzymes
  produced in this cyanobacteria.
• To use this cyanobacteria for reclamation of
  saline soil.

3

• We selected 10 organism for screening.
• Oscillatoria salina 110551.
• Oscillatoria boryana 91531.
• Oscillatoria willei 130711.
• Chrococcus minor 91342.
• Synechococcus elongatous 141741.
• Spirulina subsalsa 101022.
• Phormidium valderianum 20041
• Phormidium valderianum 30501.
• Phormidium corium 91921
• Gleocapsa crepidium 20372.

4

• Out of 10 organisms Oscillatoria willei Mo711
  tolerate high salinity.
• This organism is treated in 3 different
  conditions 0,25,100 ppt.
• The treatment is done for 24 , 48 hours.
• Then it is centrifuged protein is extracted.
• protein is estimated and loaded in native gel for
  anti-oxidative enzyme study.

5
Anti oxidative Enzyme

• SOD staining
• Gel is washed in Tris HCl PH 8.
• Riboflavin - 4mg, EDTA - 2mg, NBT 20mg are
  added to Tris HCl PH 8.
• Incubate gel for 30 min in dark.
• Illuminate the gel under light for 10- 15 min at
  RT.
• Achromatic regions are visualised dark blue
  background.

6
Total SOD
24 Hrs 48 Hrs
0 25 100 0 25 100
7
Types of SOD
24 Hrs 48 Hrs
24 Hrs 48 Hrs
0 25 100 0 25 100
0 25 100 0 25 100
8
Peroxidase

• 3,3-diaminobenzidine(DAB) oxidised DAB.
• Gel is washed with sodium acetate buffer PH 4.5.
• DAB 30mg, H202 - 250µl are added to sodium
  acetate buffer incubate gel in this solution
  until brown bands are visualised.

9
Peroxidase
24 Hrs 48 Hrs
0 25 100 0 25 100
10
Catalase

• H2O2 H2O O2
• Gel is washed in potassium phosphate buffer PH
  7.0.
• Gel is incubated in potassium phosphate buffer
  containing H2O2 ?3µl, 2 potassium ferric
  cyanide, 2 ferric chloride.
• The yellow colour bands are appeared on a dark
  green background.

11
Esterase

• Gel is washed in phosphate buffer pH 7.5.
• Gel is incubated in phosphate buffer PH 7.5
  containig a- napthyl acetate ? 50mg, ß -napthyl
  acetate ? 50mg,Fast blue RR ? 100mg .
• Incubate the gel in this solution until dark gray
  colour band appeared.

12
24 Hrs 48 Hrs
0 25 100 0 25 100
13

• SDS PAGE still in process.
• We are standardizing the SDS-PAGE.
• Yet to be done
• 2D gel

14
SDS-PAGE
24 Hrs 48 Hrs
0 25 100 0 25 100
15
(No Transcript)