Pre lab preparations:

1. Stock antigen = 25~g/500~1=0.05 ug/ul =50 ug/mL. Calculate the antigen volume that need to
run all the protocol and dilute the stock to become a working concentration at 1 ug/mL.
2. Stock of primary antibody = 25~g/500~1=0.05 ~g/1l1 =50 ug/mL. Calculate the volume that need
to run all the protocol and dilute the stock to become a working concentration at 1 ug/mL.
3. Stock of secondary antibody = 25~g/500~1=0.05 ~g/~l =50 ug/mL. Calculate the volume needed
to run all the protocol and dilute the stock to become a working concentration at 1 ug/mL.
4. Prepare 0.18M H2S04 from 98.097g/mol of H2S04.

Antigen: Chicken gamma globulin

Primary antibody: Polyclonal anti-chicken antibody made by rabbits
Secondary antibody: Polyclonal anti-rabbit antibody made by goats linked (conjugated) to horseradish
peroxidase (HRP)
Enzyme substrate: 3,3',5,5'-tetramethylbenzidlne (TMB) - a colorless solution that when oxidized by
HRP turns blue

1. Antigen
- Protocol I: 50µl x (0+1) x 3 = 150 µl
- Protocol II: 50 µl x (0+1) x 2 = 100 µl
- Protocol III:50 µl x (3+2) x 2 = 500 µl
- Protocol IV: i) 50 µl x (0+1) x 3 = 150 µl
ii)50 µl x (0+1) x 3 = 150 µl
- Total Antigen volume required: 1050 µl
-Volume required to dilute stock antigen from 50µg/ml to a working concentration of 1µg/ml
M1V1 = M2V2
(50µg/ml) V1 = (1µg/ml) (1050µl)
VI = (1µg/ml) (1050 µl)
(50µg/ml)

V1 = 21 µl

⸫ 21 µl stock antigen(50µg/ml) + 1029µl PBS

2. Primary antibody:
- Protocol I: 50µl x (11+2) x 3 = 1950 µl
- Protocol II: 50 µl x (2+2) x 2 = 400 µl
- Protocol III:50 µl x (0+1) x 2 = 100 µl
- Protocol IV: i) 50 µl x (12+2) x 3 = 2100 µl
ii)50 µl x (5+2) x 3 = 1050 µl
- Total Primary Antibody volume required: 5600 µl
-Volume required to dilute stock Primary Antibody from 50mg/ml to a working concentration of
1mg/ml
M1V1 = M2V2
(50µg/ml) V1 = (1µg/ml) (5600 µl)
VI = (1µg/ml) (5600 µl)
(50µg/ml)

V1 = 112 µl
⸫ 112 µl stock antigen(50µg/ml) + 5488µl wash buffer

3. Secondary antibody:
- Protocol I: 50µl x (11+2) x 3 = 1950 µl
- Protocol II: 50 µl x (2+2) x 2 = 400 µl
- Protocol III:50 µl x (3+2) x 2 = 500 µl
- Protocol IV: i) 50 µl x (12+2) x 3 = 2100 µl
ii)50 µl x (5+2) x 3 = 1050 µl
- Total Secondary antibody volume required: 6000 µl
-Volume required to dilute Secondary antibody from 50µg/ml to a working concentration of 1µg/ml
M1V1 = M2V2
(50µg/ml) V1 = (1µg/ml) (6000 µl)
VI = (1µg/ml) (6000 µl)
(50µg/ml)

V1 = 120 µl
⸫ 120 µl Secondary antibody (50µg/ml) + 5880µl wash buffer

4. Volume of 0.18M H2S04 to be used for termination of enzymatic reactions:

- Protocol IV: i) 25 µl x (12+2) x 3 = 1050 µl
ii)25 µl x (5+2) x 3 = 525 µl
- Total 0.18M H2S04 volume required: 1575 µl
 Prepare 0.18M H2S04 from 98.097g/mol of H2S04:

Molarity of H2S04 ?
- 98.097g/mol
- Density of H2S04: 1.84g/mL  1840g/L
- 1840g : 98.097g
L mol
- 1840 mol
98.097 L
- ⸫ Molarity H2S04 = 18.76M

- M1V1 = M2V2
(18.76M) V1 = (0.18M) (1575µl)
VI = (0.18M) (1575µl)
(18.76M)

V1 = 15.11 µl ~15µl

- 15µl of 98.097g/mol of H2S04 + 1560 µl distilled water