Protocol for Total Protein Determination -Lowry et
al., 1951 Last
updated 4/9/2012 Sitti
REAGENT C
2% Na2CO3 in 0.1N
NaOH - 50 ml
0.5% CuSO4.5H20 in 1%
Na Tartrate - 1 ml
REAGENT E
Diluted
1N Folin-Ciocalteu phenol reagent
PROTOCOL
1. Filter
10 ml aliquot/culture onto pre-combusted GF/F filter (fold and wrap in
aluminium foil - store at -80 oC
/LNG if not analyze) *make 3 replicates for each PBR tube.
2. Homogenize
filter in glass-teflon homogenizer in 3 mL 1N NaOH. Rinse 2-3 times homogenizer
with 1N NaOH and collect as maximum extract as possible in a falcon tube, bring
up the volume to 10 ml and cap tightly.
3. Keep
the extract at room temperature for 22-24 hours for complete extraction.
4. Vortex
for thorough mix and centrifuge the extract for 5 mins at 5000 rpm.
5. Take
0.5 mL of extract and mix with 5 mL of
freshly prepared reagent C.
6. Allow
to stand for 10 min. (in dark)
7. Add 0.5
mL freshly prepared reagent E, mixed well.
8. Allow
to stand for 30 minutes to develop maximum colour. (in dark)
9. Read
absorbance at 750 nm against a reagent blank.
10. Use Bovine
serum albumin prepared in 1N NaOH as standard for total protein contents
determination. To get the standard
curves or calibration curves, prepare standard solution at concentration 0 - 150
µg/µl
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