Preparation of polyclonal antibodies against phosphopeptides.

Summary

It is possible to prepare anti-phosphopeptide antibodies (i.e., antibodies that recognize phosphorylated peptides) that recognize only proteins in the phosphorylated state and do not cross-react with non-phosphorylated similar proteins or other phosphoproteins. Since phosphorylation often implies that a protein is in a functional or active state, this antibody is a convenient probe of the functional state of a protein. Unlike conventional antibodies, anti-phosphopeptide antibodies provide information not only about the content of the protein but also about its activity. Purification of polyclonal antibodies involves multi-step affinity chromatography to negatively screen and positively screen antibodies with suitable reactivity. Sourced from Compact Molecular Biology Laboratory Guide (5th Edition)

Operation method

basic program

Materials and Instruments

Crude serum from rabbits immunized with phosphopeptide-BSA crosslinks
PBS/azide MgCl2
Spectrophotometer Dialysis bag Columns filled with BSA-agarose affinity medium Columns filled with phosphotyrosine affinity medium Columns filled with homologous non-phosphopeptide affinity medium Columns filled with homologous phosphopeptide affinity medium Columns filled with positive-screening phosphopeptide affinity medium

Move

1. connect the column filled with BSA-Agarose Affinity Medium and the column filled with Tyrosine Phosphate Affinity Medium in tandem.

2. Allow approximately 15 ml of crude serum to flow by gravity through both columns. Wash with PBS/azide solution until all the yellow serum passes through the columns (or detect the _A280 of the effluent spectrophotometrically until it reaches the basal absorbance value), and then wash with 5-10 ml of PBS/azide solution to collect all the washings that pass through the columns, which may contain the antibody of interest.

After the serum has been passed through the column, the column can be regenerated by washing it sequentially with 10 times the volume of the column bed with 3 mol/L NaSCN and 10 times the volume of the column bed with PBS/azide solution, and then storing it in the PBS/azide solution at 4°C until ready for use. 3.

3. Allow the post-column serum to flow by gravity through the column filled with the non-phosphopeptide affinity medium several times to remove as much cross-reactivity as possible, and sequentially wash the column with 10 times the column bed volume of 3 mol/L NaSCN and 10 times the column bed volume of PBS/azide solution to regenerate the column between each pass.

4. if cross-reactivity with homologous phosphoproteins is expected, flow the serum from the previous step through the column filled with homologous phosphopeptide affinity medium using the method in Step 3.

5. Pre-moisten and wash a dialysis bag approximately 25 cm long before collecting the positive-screening affinity purified fraction. Clamp one end with a dialysis clip and check for leaks. Also pre-prepare 6 L of PBS/azide dialysate and cool to 4 °C. 6.

6. Flow the serum from the previous chromatographic pass through the Positive-Screening Phosphopeptide Affinity Column three times without washing the column after each pass.

7. Collect the serum from the last pass through the column and wash the column with 5-20 ml of PBS/azide solution (amount depends on pre-column volume and column bed volume), combining the wash solution with the serum from the pass through the column. The column is then washed with 20 ml of PBS/azide solution and the wash solution is collected separately as a "wash" fraction. 8.

8. Elute with the following leaving agents of choice: 20 ml of 3 mol/L NaSCN, or 10 ml of 3.5 mol/L MgCl₂ and 10 ml of 4.5 mol/L MgCl₂, in that order. Immediately after starting the elution, collect approximately 3 ml of the elution fractions and place them directly into the dialysis bag prepared in step 5, hold the bag proximal to the bag with a bag clip and immediately place it into the PBS/azide dialysate. dialysate immediately. Collect at least 6 elution fractions. 9.

9. Dialyze the PBS/azide solution at 4 °C with the elution fractions collected in step 8. 10.

10. The protein yield in the dialysed fraction is measured and calculated by measuring the absorbance at 280 nm or by protein colorimetry. Typically > 1 mg of purified antibody is obtained from 15 ml of serum sample. 11.

11. Dispense the antibody into small portions and store at -70°C; store at 4°C as a working solution.

12. Detect reactivity and cross-reactivity of the final sample and the fractions collected in the previous steps by ELISA and immunoblotting.

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