CLx328 – Biologics Purification


Lab 4 – Purification of IgG

Procedures

Equilibration of Protein A Column





1. Loosen Top Cap and Remove/Snap off bottom cap



2. Place it in a 15 mL collection tube and centrifuge for 1 minute at 1000 g.



3. Equilibrate the spin column by adding 2 mL of binding buffer. Centrifuge for another 1 minute and discard flow-through.



4. Repeat step 3.


                                                        CLx328 – Biologics Purification

Addition of Sample to Column

1. Cap bottom of the spin column with the rubber cap.

2. Add 1 mL of Serum followed by another 1 mL of binding buffer into the spin column.

3. Cap the top of the spin column and incubate at room temperature with end-to-end mixing for 10 minutes.

4. Loosen top cap and remove bottom cap of the spin column.

5. Place the spin column in a new 15 mL collection tube and centrifuge for 1 minute. Collect the flow-through.

6. Set the UV Spectrometer wave length to 280nm.

7. Place the spin column into a new 15 mL collection tube and wash by adding 2 mL of binding buffer by centrifuging for 1 minute. Collect flow-through.

8. Transfer the collected flow-through into a cuvette.

9. Using KIM wipes, wipe the clear side of the cuvette clean for the UV Light to pass through.



10. Place the cuvette into the UV Spectrometer, with the clear side facing the direction where the UV light will shine through

11. Press "Read Cuvette" to shine the UV light through the cuvette. Record your readings.



12. Repeat steps 7 - 11 until the UV Absorbance of the flow-through is close to that of the binding buffer.



                                                        CLx328 – Biologics Purification


Elution of Antibody

1. Add 100 µl of neutralization buffer to 5 new 15 mL collection tubes and place the spin column into one of the tubes.

2. Add 1 mL elution buffer to the column and centrifuge for 1 minute into the first of the five collection tubes containing the neutralization buffer. Collect the flow through as the first Elution fraction.


3. Repeat step 2 to obtain a total of 5 different fractions.







4. Determine which fractions contain the purified antibody by measuring the relative absorbance of each fraction at 280nm using the UV Spectrometer.