Objective : Optimization of conjugation condition (Titration) for Protein A

Date :

1) INTRODUCTION
This document details the procedure for optimizing the conditions for conjugation of a Protein A to DCNovations Colloidal Gold by titrating the colloidal
gold pH vs. protein A concentration.

2) scope
The results provided by this procedure identify the optimal pH and protein A concentration for the production of a stable protein A gold conjugate.

3) Materials and equipments required

Item Make Cat. No. Lot number

DCNovations colloidal Gold

0.2 M potassium carbonate

10% sodium chloride
Protein A (0.1 mg/ml
soluntion prepared in
water)

Other Materials required

Beaker, teflon stir bar, micropipette, plastic cuvette, spectrophotometer, pH meter, glass test tubes

4) saftey
Follow general laboratory safety precautions.
Wear appropriate personal protective equipment including lab coat, gloves.
5 ) Procedure
Note: Protein A should be dialyzed into 10mM phosphate, or an equivalent salt free buffer if the protein contains salt in its buffer.
a) pH the appropriate volume of colloidal gold.
Note- The number of pH levels and protein A concentration being tested can vary depending on the volume of material for optimization.
b) detemine the total volume of colloidal gold required at each pH level
As per DCN Gold conjugation protocol

Total
Total vol of volume of Actual
Protein A
volume of gold per tube OD 1 Gold Total no. of OD 1 Gold volume of OD
x concentratio = x =
(ml) required/pH pH levels per total no. 1 Gold taken
ns
level (ml) of pH levels (ml)
(ml)

0.52 x 9 = 4.68 x 5 = 23.4

c) Adjust the pH of the colloidal gold with 0.2M potassium carbonate and record the actual pH values on the chart below
pH level to be tested pH 5.5 pH 6.0 pH 6.5 pH 7.0 pH 7.5
Actual pH
d) Arrange and label glass test tubes according to the grid below
Desired
Desired protein A conc
protein A pH pH pH pH pH
(mg/ml)
conc (ug/ml)
0.01 10 5.5 6 6.5 7 7.5
0.008 8 5.5 6 6.5 7 7.5
0.004 4 5.5 6 6.5 7 7.5
0.002 2 5.5 6 6.5 7 7.5
0.001 1 5.5 6 6.5 7 7.5
0.0008 0.8 5.5 6 6.5 7 7.5
0.0006 0.6 5.5 6 6.5 7 7.5
0.0004 0.4 5.5 6 6.5 7 7.5
0.0002 0.2 5.5 6 6.5 7 7.5
e) Pipette exactly 0.5ml of colloidal gold at the appropriate pH into the glass test tubes.
f) Calculate the volume of Protein A required for each concentration
Desired Required Protein A Volume of
Volume of
Volume of colloidal gold protein A Amount of stock protein
x = ÷ = protein
(mL) conc protein A conc(mg/ml required in
required(ml)
(mg/ml) (mg) ) (µl)
0.5 x 0.01 = 0.005 ÷ 0.1 = 0.05 50
0.5 x 0.008 = 0.004 ÷ 0.1 = 0.04 40
0.5 x 0.004 = 0.002 ÷ 0.1 = 0.02 20
0.5 x 0.002 = 0.001 ÷ 0.1 = 0.01 10
0.5 x 0.001 = 0.0005 ÷ 0.1 = 0.005 5
0.5 x 0.0008 = 0.0004 ÷ 0.1 = 0.004 4
0.5 x 0.0006 = 0.0003 ÷ 0.1 = 0.003 3
0.5 x 0.0004 = 0.0002 ÷ 0.1 = 0.002 2
0.5 x 0.0002 = 0.0001 ÷ 0.1 = 0.001 1

g) Pipette the calculated volumes of protein into the test tubes, respectively. Gently agitate the tubes then let stir for 5 minutes at room temperature.
NOTE: No color change or aggregation should occur with the addition of the antibody. If the colloidal gold aggregates or turns purple, the protein A may
need to be dialyzed.

Colour change observation if any :

h) Pipette 100ul of 10% NaCl into each tube. Gently agitate then let sit for 5 minutes at room temperature. Scan all of the gold solutions between 400-
700nm. Additionally, scan a sample of unconjugated gold at OD1. Record the peak absorbance and peak wavelength for each sample in the table below.

pH 5.5 pH 6.0 pH 6.5 pH 7.0 pH 7.5

Peak OD
Peak OD Peak OD Peak OD
Measure & record peak Peak Peak Peak (Abs.) & Peak Peak OD Peak
(Abs.) & (Abs.) & (Abs.) &
OD & wavelength wavelength wavelength wavelength color wavelength (Abs.) & color wavelengt
color change color change color change
(nm) (nm) (nm) change (nm) change (Y/N) h (nm)
(Y/N) (Y/N) (Y/N)
(Y/N)
0.01 mg/ml
0.008 mg/ml
0.004 mg/ml
0.002 mg/ml
0.001 mg/ml
0.0008 mg/ml
0.0006 mg/ml
0.0004 mg/ml
0.0002 mg/ml
unconjugated
i) for conjugation select the condition that produced peak wavelength and absorbance values most similar to the unconjugated gold
note- There may be more than one pH within this range in which case select the configuration that requies the least amount of Protein A.
Observation:

Conclusion

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