m gram peeled

pineapple homogenize the pineapple juice

peeled pineapple

6000 rpm
15C
10 mins collect the
supernatant

Solution I
crude bromelain
extract
 cool 30mins

50ml solution I
Solution I V=50ml + 200ml ethanol

Solution I

Determine the protein

concentration by Bradford compaire the activity
of solution I and II
Determine bromelain
activity by Muruchi

precipitant Solution II
Purification fold
+ 10ml buffer Solution II
 Bradford Protein Assay

0 1 2 3 4 5 Vol of 0,1 mg/ml BSA (ml)

10 9 8 7 6 5 Deionized water (ml)

0 10 20 30 40 50 Protein concentration ( 15mins

pipet 0,5ml

2,5ml Bradford

Solution I Solution II Samples

Calculate concentration
Calbrication curve of sample

595nm
 Bromelain Assay
10mins

Filtration
2,5ml

(1) (2) (3)

Tyrosine Sample Blank
(1) (2) (3)

620nm
2ml 1N NAOH +
0,5ml Folin reagent Bromalein activity
Shake well = (Ul/ml)
Wait for 15mins

Specific activity
=
 set up
pour gel the plate

spacers
solution I, II
Glass pleates
 Loading buffer 5l sample
into well
Mix 10 Boil 10mins

Bromophenol reaches the

bottom of resolving gel Remove the gel from glass plates Stain gel with
Mark orientation of gel Coomassive Blue
Turn off equipment

Wash gel with Visualize the band

10% acetic acid