Transcript ESTIMATION OF AMINO GROUPS USING TNBS

ESTIMATION OF AMINO
GROUPS USING TNBS
REFERENCE: Fields, R. 1972. Methods in
Enzymology. 25:464-469.
Kris-itd.unair.ac.id (for education purpose only)
MATERIALS :
Solution A: 100mls of 0.1M Na2SO3 (fresh each week)
Solution B: 1.0l of 0.1M NaH2PO4
Solution C: 1.0l of 0.1M Na2B4O7 in 0.1M NaOH (make up in acid
and ddH2O- washed glass).
Trinitrobenzene sulfonate (TNBS) 10g in 10ml H2O, heat to
dissolve and remove black flecks of oil by centrifugation. Add HCl
to 2M and cool to room temp. Wash the crystalline precipitate on
a glass filter with 1M HCl. Desiccate and store at 4oC in brown
bottle. Make up to 1.1M fresh daily (100mg recrystallized TNBS in
0.2ml H2O).
METHOD :
1. make fresh daily Solution D: 1.5ml Solution A + 98.5ml
Solution B
2. standard curve: BSA at 0.1, 0.2, 0.5, 1.0, and 2.0 mg/ml in
0.25ml H2O.
3. samples: same concentration range as above in same
volume.
4. add 0.25ml solution C
5. add 10µl TNBS (take note of time!)
6. Incubate exactly 5 min at 23oC.
7. add 1ml solution D to stop reaction.
8. measure OD420. Standard curve should range from about
0.09 to >1.8
CALCULATION :
OD420 = 1.0 = 52nmol amino groups = 78nmol/1.5ml assay mix
"1.0mg/ml" BSA sample (0.25mg) = OD420 of 0.945 = 73.41nmol
NH3
therefore, 0.25 mmol BSA = 73.41 nmol NH3 groups
67,000
3.73nmol BSA = 73.41 nmol NH3
therefore 1 molecule BSA = 19.8 molecules of lysine.