Editing Antibody Purification

1. Dilute serum with 3 volumes of loading buffer. 

<br>2. Apply diluted material to column. Collect unbound fraction in a test tube or beaker and save for possible reprocessing. 

<br>3. Wash unbound proteins with 5 column volumn loading buffer.

<br>4. Apply Elution Buffer to column. Collect fractions equal to 1/2 column volume. Use 10 ml elution buffer per ml resin.

<br>5. Re-equilibrate the column with 5 ml loading buffer per ml resin. Check pH of effluent to ensure that column is equilibrated at the pH of the loading buffer.

<br>6. Assay eluate fractions for protein by absorbance at 280 nm. 

<br>7. Pool fractions which are positive for protein. Neutralize to pH 6 - 8 with 1.5 M Tris base.

<br>8. If desired, the unbound fraction may be re-applied to the column to recover any Ig that did not bind on the first pass, which may occur if the amount of material loaded exceeds the column capacity.

<br>9. Desalt by FPLC 

or dialyze into desired buffer, i.e. PBS, pH 7, at 4 °C. The volume of the dialysis buffer should be at least 20 times the volume of the protein solution. At least 2 changes of dialysis buffer, for at least 2-4 hours each, should be done to ensure complete equilibration in the dialysis buffer.

<br>10. If no more runs are to be performed, wash the column with PBS containing 0.05 - 0.1% sodium azide. Seal the column with a stopper or Parafilm and store at 4 °C. 

Protein A Loading Buffer: 1 M potassium phosphate, pH 9.0<br>Protein G or L Loading Buffer: 0.01M phosphate buffered saline (PBS), pH 7.4.<br>Elution Buffer: 0.1 M citric acid, pH 3.0<br>1.5 M Tris base, for neutralization of the eluate 

Rabbit: 12 - 14.5mg IgG/ml serum.<br>Human: 7.5 - 22 mg IgG/ml serum<br>Rat: 5 - 7 mg IgG/ml serum<br>Mouse: 2 - 5 mg IgG/ml serum<br>Goat: 18 - 24 mg IgG/ml serum<br> 

Table 1. Affinity of Protein A for IgG subclasses. <tab border="1">
Species	Subclass	Binding pH	Elution pH
Mouse	IgG1	8.5-9.0	6.0-7.0
Mouse	IgG2a	8.0-9.0	4.5-5.5
Mouse	IgG2b	8.0-9.0	3.5-4.5
Mouse	IgG3	8.0-9.0	4.0-7.5
Rat	IgG1	8.0-9.0	6.0-8.0
Rat	IgG2a	9.0	7.5-9.0
Rat	IgG2b	8.0-9.0	7.0-8.0
Rat	IgG2c	8.0-9.0	3.0-7.0
Human	IgG1	7.0-7.5*	2.5-4.5
Human	IgG2	7.0-7.5*	2.5-4.5
Human	IgG3	7.0-7.5*	3.0-7.0
Human	IgG4	7.0-7.5*	2.5-4.5
Rabbit	IgG	7.5	3.0-7.0
Guinea pig	IgG1	7.5-9.0	4.0-5.0
Guinea pig	IgG2	7.5-8.0	3.0-4.5
</tab> 

<br> 

'''Protocol 2'''<br> 

'''this is for 1 ml protein A beads'''<br> 

Affinity Purifying Antibody from Rabbit Serum 

If your antigen was a fusion protein then you will usually need to purify your antibody in two steps. The first step is to run the serum over a column containing the fusion partner (usually GST in our lab but many other fusion partners are used). This step removes the antibodies that recognize the fusion partner. Obviously if your fusion partner was Protein A or Protein G you cannot do this step as your specific antibody will also bind to Protein A. You can use Protein L as rabbit antibodies bind poorly to Protein L.<br>The second column contains beads coupled to the fusion protein that you used as antigen. The antibodies you want flow-through the first column and stick to the second column. Because they are so similar, the instructions here are for the second column. 

1. Wash Sepharose beads that have antigen coupled to them as follows:<br>&nbsp;&nbsp;&nbsp;&nbsp; 1.1. 3X PBS with 1mM PMSF and 1X PIN<br>&nbsp;&nbsp;&nbsp;&nbsp; 1.2. 2X with 0.2M glycine pH 2.65 (to get rid of stuff on the columns)<br>&nbsp;&nbsp;&nbsp;&nbsp; 1.3. 2X with 0.1M NaHCO3 / 0.5 NaCl pH 8.3 + PIN + PMSF<br>&nbsp;&nbsp;&nbsp;&nbsp; 1.4. 2X with PBS + PMSF +PIN<br> 2. Check pH and adjust to approx. 7.5 if necessary

<br> 3. Incubate 0.5 ml beads with 3ml serum + 4ml PBS, overnight at 4°C.

<br> 4. Pour beads into column and let settle.

<br> 5. Collect the flow through (will contain unbound proteins from serum).

<br> 6. Wash twice with 10mL of PBS, until OD280 ~= or &lt; 0.05

<br> 7. Elute bound antibodies with batch of 1.5 mL of 0.2M glycine pH 2.6. Collect 400 uL per fraction into 1.5mL tubes containing 120 uL of 1M Tris pH 8.0. This neutralizes the glycine buffer, until OD280 ~= or &lt; 0.05.

<br> 8. Do a Bradford assay or OD280 to check for protein peaks.

<br> 9. Run a gel for the peak fractions to make sure that they contain only antibody and then combine these fractions. Test antibody. It is often a good idea to dialyze the buffer down to 50 mM Tris HCl pH 8.0, add 50% glycerol and store at -20°C.<br> 

[[Category:Protocols]]