Tuition cancelled....NEVERMIND!

Wha... I thought I would go off earlier for tuition by starting my eperiment earlier today. In the end, tuition canceled. Also good la. The weather's getting abnormal these days. Precipitation to the MAX!

So early in the morning, I was the only person in the lab.. Slogging through the ELISA expt Slapping plates, pipetting solution. Then See Jye come and disurb me again... say what I don't know how to switch on the light... hey! excuse me. I just dun wanna press wrong button and cause an emergency ma. Then I went on to do my cell passaging. Looks like my cells are pretty strong. 3 deaths out of 255. Pretty cool. Like Tamagotchi like that.

At the same time, I was doing stupid pipette tip refilling...... really testing my patience man. Qian Qian must be damn happy la.. don't have to do duty. I still have to come back this Saturday to topup media ok! This is called responsibility.

Then in the afternoon, the Elisa expt still proceeds but at the same time I went to prepare media for the cells... running out le. Dr ng is so nice to show me how to prepare them. Then Jia Juan and Loo ling both said that I look damn busy and were seeing me practically all around the place.

Ok.. The moment came where I pray for justice of my hard work.. The result time! Iwas dancing infront of the computer, hoping the results were consistent. YEAH! The results were quite spectacular man...


ELISA
Date of expt: 13/03/2008
Plate:
1 Strip
8 Wells in total
Well no.
Absorbance
1
2.6668
2
3.289
3
3.7831
4
0.1291
5
3.1208
6
3.8632
7
3.6976
8
0.1018

Lvl 4 Absorbance reader machine
Wavelength:2
Reading at: 450nm
Reference at: 630nm
Parameters/ Variables:
a) Capture Ab : 250 ng/ml, Overnight incubation at 4 Deg
b) Standard for detection : Biolegend, 4h incubation room temp
Concentrations (pg/ml): 1000, 0 (for control)
c) Detection Ab : 2 ug/ml, 1ug/ml, 0.5ug/ml, 1h incubation
d) Avidin-HRP : 1:2000 dilution, 1h incubation
e) TMB: ready to use, 30 min incubation
f) TMB stop solution (0.5M Sulphuric Acid)

1
2.5377

2
3.1599

3
3.654
0.5 ug/ml
1.76885
5
3.019
1 ug/ml
2.57995
6
3.7614
2 ug/ml
3.327
7
3.5958


Readings with negative controls deducted



1, 5-Row containing 0.5 ug/ml of Antibody detection
2, 6- Row containing 1 ug/ml of Antibody detection
3, 7- Row containing 2 ug/ml of Antibody detection


--------ok a snippet of some of the data..

Now for some Hardcore stuff:

Protocol : ELISA for Human IFN-γ
Aim: To optimize concentration of antibody capture, detection used, and the optimal incubation time/temperature
1. Media Perparation
(a)Wash Buffer 2L, PBS/0.1% Tween20
-2L MiliQ water+10 Tablets of PBS+2ml Tween20
-Stir 2-3 hrs, no need to filter
(b)Dilution solution 400ml, PBS/1%BSA
-400ml MiliQ water+2 Tablets PBS+ 4g BSA
-Stir 2-3 hrs before filter
(c)Blocking solution 400ml,PBS/3%BSA
-400ml MiliQ water+2 Tablets PBS+ 12g BSA
-Stir 2-3 hrs before filter
(d)PBS Solution
-400ml MiliQ water+2 Tablets PBS
(e)TMB stop solution
-Prepare 2M sulphuric acid by dilution from stock at media prep room (level 3)

2. Antibody capture
-Coat 96 well plates (Nunc Maxisorp) with 100ul of purified Mouse monoclonal [GIF-3] to Interferon gamma (abcam ab9801) to each well in use (total 24)
-Stock solution is 2mg/ml
-Dilute with PBS 1/1000 to 1/10000 times. Prepare 3 sets of Antibody capture with different concentrations. Preferably the concentrations: 2000ng/ml, 1000 ng/ml, 250 ng/ml
-Do a X1000 dilution with stock solution using PBS solution to obtain 2000ng/ml followed by serial dilutions:
for 2000ng/ml: 2uL Stock+1998uL PBS
for 1000ng/ml: 500uL of 1.+500uL PBS
for 250ng/ml: 200uL of 2.+600uL PBS
- Seal plate and incubate it overnight at 4 Deg Celsius
-Flick off unbound antigen and wash well 3 times (300uL each well) with PBS/0.1%Tween20 Wash buffer. During each wash, tap the plate after adding wash buffer to evenly distribute the wash. Then, invert the plate onto a dry piece of tissue paper to remove residual wash solution
3.Blocking
-Add 300ul of blocking solution to all wells in use and incubate it at room temp for 1 hour
-Then wash 3 times with PBS/0.1%Tween20 (wash buffer) and tap the inverted plate onto a dry piece of tissue paper to remove residual wash solution
4. Antibody Binding (standards)
-Recombinant Human IFN-gamma have been diluted from stock conc. of 0.05mg/ml to 1ug/ml (1:50 dilution prepared by Lina)
-Prepare in total, 20 sets of standards of the same concentration of 2000pg/ml. Hence, total volume should be 2ml.
-Do a dilution of 1:500 on Lina’s Standards by mixing 4uL of Standard with 1.996ml of 1%BSA/PBS to get a concentration of 2000pg/ml.
-These standards serve as a basis for the materialization of the standard curve
-Add 100ul of diluted standards to each well Except for the last row, which is to be used as control
-For negative controls, 100ul of PBS/1%BSA is added
-Incubate for 2-4 hours at room temp
-Invert, wash 3 times with PBS/0.1%Tween and tap the inverted plate onto a dry piece of tissue paper to remove residual wash solution
5. Antibody detection
-Dilute Mouse monoclonal [4S.B3] to interferon gamma (Biotin), ab21538 from stock concentration of 0.5mg/ml to 1ug/ml (6uL stock+2.994mL diluent=2.5mL Total)
-Dilute with 1%BSA/PBS
-Add 100ul of diluted antibody to each well
-Incubate for 1 hour at room temp
- Invert, wash 3 times with PBS/0.1%Tween and tap the inverted plate onto a dry piece of tissue paper to remove residual wash solution
6. Enzyme attachment
-Dilute X2000 of Avidin-HRP conjugated (BioLegend, #405103) with 1%BSA/PBS
-Add 100ul of diluted enzyme to all wells
-Incubate at room temp for 30 mins
- Invert, wash 5 times with PBS/0.1%Tween and tap the inverted plate onto a dry piece of tissue paper to remove residual wash solution
7. Enzyme Detection via Substrate
-Add 100ul of readymade Sigma TMB solution (T0440) to all wells
-Incubate at room temperature for 3 times of 15 mins interval (total reaction time about 45mins for last two columns of wells)
8. Measurement
-At the end of each 15 mins interval, stop the reaction for one column of wells and its duplicate (see diagram 1) for absorbance reading
-Add 100uL of TMB stop solution (2M Sulphuric Acid) to one column of wells and its duplicate and incubate for 15 mins before adding TMB stop to the next two columns of wells
-Use ELISA Machine (@level 4) to read absorbance at 450nm, reference 630nm, with wavelength 2 of the columns of wells and its duplicates
-Determine the average absorbance of each standard with their duplicates and subtract absorbance value with that of the negative control
9. Standard curve
-Plot Reaction time. VS Absorbance (OD) to obtain curve for each of the 3 different antibody capture concentration
-The lowest reaction time will have the lowest absorbance
-Choose the one (antibody capture conc) with the highest absorbance
10. Further exploration
-Repeat the ELISA experiment, this time varying incubation times, antibody capture concentrations (0.5ug/mL-2.0ug/mL) and standard concentrations

------- This was my home work ok..
I had to devise my own experiment with the parameters given!

Actually damn proud of myself. Because I actually don't really like Biology but I hapen to be so immersed in the area of research.

Ok.. I owe Janice some work today due to my busy expt schedule. Shall prepare some solutions tomorrow.

oh yes.. Tonight is home alone with Mummy. Despite her bad body ache and pains, she still cook a splendid dinner for me! She even gave the whole piece of red snaper steak to me. See.... parents really do love us! Today I really gave my heart into massaging my mum. Noticing her unbearable pain really made my heart sank.. I love you mummy. I will pray for you!

Muackz.. Good Night!