TNBSA Assay
September 10, 2016
Present: Lily, Tyra
Task: Assay Prep Work
- Made 0.01% solution of TNBSA: added 10 uL of picrylsulfonic acid solution (5%) to 4990 uL dH2O
September 13, 2016
Present: Lily, Tyra
Task: TNBSA Assay Run 1
DSPE-PEG-CONHNH2 Sample Prep:
- 40 ug/mL: Added 16 uL DPC + 484 uL Sodium Bicarbonate
- 160 ug/mL: 64 uL DPC + 436 uL Sodium Bicarb
- 400 ug/mL: 160 uL DPC + 340 uL Sodium Bicarb
- Note: original concentration of DSPE-PEG-CONHNH2 estimated to be 1.25 mg/mL (may have experiences some loss over carbohydrazine synthesis steps
Amino Acid Standard Calculations (used Cysteine not Glycine)
- Made stock of 0.1 mg/mL
- Weight of tube: 0.8902 g
- Weight of cysteine: 0.6 mg
- 60 ug/mL: 300 uL stock + 200 uL sodium bicarb
- 20 ug/mL: 100 uL stock + 400 uL sodium bicarb
- 10 ug/mL: 50 uL stock + 450 uL sodium bicarb
- 2 ug/mL: 10 uL stock + 490 uL sodium bicarb
- 0.2 ug/mL: 1 uL stock + 499 uL sodium bicarb
Plate 1: * used columns 9-12
A9 – A12: DPC 400
B9/B10: DPC 160
B11/B12: DPC 40
C9/C10: DPC 160
C11/C12: DPC 40
D9 – D12 Cysteine 60
E9 – E12 Cysteine 20
F9 – F 12 Cysteine 10
G9 – G12 Cysteine 2
H9 – H12 Cysteine 0.2
At 60 uG/mL standard curve was not following linear trend, we’re going to modify the concentrations used tomorrow and go up to Max 30. We will also use glycine, not cysteine.
Trial 2
September 14, 2016
Present: Lily
Task: TNBSA Assay Run 2
- Made the following changes to protocol since yesterday:
- Prepared glycine standards at concentrations 0 ug/mL, 5, 10, 15, 20, 30
- Stock glycine preparation:
- tube weight: 893.3 mg
- glycine added: 1.0 mg
- 15 mL stock TNBSA preparation:
- Added 30 uL picrylsulfonic acid solution (5%) to 15970 uL milliQ dH2O
Plate 1:
A1 – A6: Blank
A7 – A12: Glycine 5
B1 – B6: Glycine 10
B7 – B12: Glycine 15
C1 – C6: Glycine 20
C7 – C12: Glycine 30
D1 – D6: DPC 40
D7 – D12: DPC 160
E1 – E6: DPC 400
Sept 21, 2016 Present: Ina
Task: Make a 1.33 mM stock solution of Glycine in Sodium Bicarbonate
note: made 2 batches of stock solution of the same 1.33 mM concentration!
- First batch
- Weighed 2.1 mg of glycine in a microcentrifuge tube
- Reconstituted the glycine in around 1 mL of sodium bicarbonate
- Measured out 19 mL of sodium bicarbonate using a graduated cylinder
- Transferred 19 mL + 1 mL reconstituted glycine into a 50 mL falcon tube
- Vortexed mixture Labelled as 1.33 mM stock solution Glycine- placed on yellow tube rack
Task: Make 1mL of 30 ug/mL (400 uM) Glycine working solution
- Took out 300 µL of 1.33 mM stock Glycine solution
- Added 700 µL of Sodium Bicarbonate
- Vortexed mixture
-
Labeled as 30 µg/mL in RED marker
- Second batch
- 20 mg glycine in 200 mL sodium bicarbonate
- Made a 1 mL and a 500 µL 30 µg/mL working stock solution of Glycine
- Labeled as 30 µg/mL in BLUE marker
Sept 22, 2016
Present: Ina, Lily
Task: Make dilutions for TNBSA and start TNBSA
Dilutions for TNBSA calibration curve (from Ina’s BLUE stock):
- A: 20 µg/mL (266.4 µM) = 300.5 µL of 1.33 mM stock glycine + 1199.5 µL sodium bicarbonate = 1500 µL
- B: 15 µg/mL (199.8242 µM) = 750 µL of A + 750 µL of sodium bicarb = 1000 µL
- C: 10 µg/mL (133.2161 µM)= 666.6672 µL of B + 334 µL of sodium bicarb= 1000 µL
- D: 5 µg/mL (66.6081 µM) = 250 µL of C + 750 µL of sodium bicarb= 1000 µL
TNBSA Assay Results
Standard Curve
Glycine Calibration Curve
Concentration (ug/mL) | OD#1 | OD#2 | OD#3 | OD#4 |
0 | 0.358002 | 0.367515 | 0.344029 | 0.360469 |
5 | 0.492693 | 0.504721 | 0.502551 | 0.491116 |
10 | 0.609234 | 0.626726 | 0.605351 | 0.610412 |
15 | 0.710892 | 0.723744 | 0.699186 | 0.731787 |
20 | 0.780997 | 0.806564 | 0.790449 | 0.795282 |
25 | 0.814198 | 0.854335 | 0.829755 | 0.850705 |
30 | 0.906401 | 0.921332 | 0.6581598 | 0.867323 |
Corrected Absorbance
Concentration (ug/mL) | OD#1 | OD#2 | OD#3 | OD#4 | Mean |
0 | 0.1168513 | 0.1263643 | 0.1028783 | 0.1193183 | 0.1163531 |
5 | 0.2515423 | 0.2635703 | 0.2614003 | 0.2499653 | 0.2566196 |
10 | 0.3680833 | 0.3855753 | 0.3642003 | 0.3692613 | 0.3717801 |
15 | 0.4697413 | 0.4825933 | 0.4580353 | 0.4906363 | 0.4752516 |
20 | 0.5398463 | 0.5654133 | 0.5492983 | 0.5541313 | 0.5521723 |
25 | 0.5730473 | 0.6131843 | 0.5886043 | 0.6095543 | 0.5960976 |
30 | 0.6652503 | 0.6801813 | 0.4170091 | 0.6261723 | 0.5971533 |
Samples | Concentration (ug/mL) | Corrected | Mean | Concentration (ug/mL) |
1 | 0.350385 | 0.346831 | 0.348608 | 9.734311927 |
2 | 0.355847 | 0.352947 | 0.354397 | 9.999862385 |
3 | 0.363877 | 0.35736 | 0.3606185 | 10.28525229 |
4 | 0.386082 | 0.391529 | 0.3888055 | 11.57823394 |
5 | 0.429729 | 0.441918 | 0.4358235 | 13.73502294 |