Imject Maleimide Activated BSA Kit (Pierce)

Includes: Imject Maleimide Activated BSA

5 x 2 mg

All Conjugation and Purification Buffers

Prepacked Gel Filtration Columns

5 x 5 ml

See the instructions from the Pierce kit.

A. Conjugation Procedure

1. Reconstitute one vial of the maleimide-activated protein by adding 200 ul of ultrapure water to make a 10 mg/ml solution.

Note: Vortex mixing or heating will cause the protein to precipitate.

2. Dissolve the sulfhydryl-containing hapten in a volume of Conjugation Buffer equal to 1.0-2.5 times the volume of reconstituted activated protein. For example dissolve 2 mg of peptide in 200-500 ul of buffer and add it to the reconstituted protein. Alternatively, if the peptide is freely soluble it may be added as a solid to the protein solution.

Note: For haptens with limited solubility, DMSO may be used for solubilization. Use approximately 30% DMSO in the final conjugation solution or the carrier protein may irreversibly denature.

3. Immediately mix the peptide and activated protein and react for 2 hours at room temperature.

4. Purify the conjugate by desalting or dialysis to remove EDTA and sodium azide.

B. Conjugate Purification by Desalting

Use either desalting or dialysis to remove EDTA and sodium azide. If DMSO was used in the conjugation, add DMSO to the Purification Buffer Salts for desalting to prevent precipitation in the column; dialysis is not compatible with DMSO.

Desalting or dialysis will not separate non-conjugated protein; however, a large excess of hapten is used in this protocol, making it unlikely that non-conjugated carrier exists in significant quantity.

If the conjugate is to be used for injection within one week, PBS may be used for purification. If the conjugate will be frozen, use Purification Buffer Salts for purification, which will preserve the product during freeze-thaw cycles. If a precipitate formed during conjugation, centrifuge the precipitated material, collect the supernatant and save the precipitate. Purify only the supernatant. Combine the purified conjugate to the precipitate.

This is what I do. Use the columns provided with the kit.

1. For desalting, dissolve the contents of one bottle of Purification Buffer Salts by adding 60 ml degassed, ultrapure water

to the bottle. Store excess buffer at 4C.

2. Sequentially remove the top and bottom caps from a desalting column and allow the storage solution to drain. Use one

desalting column for each 0.5 ml of sample.

3. Wash column with 3-5 column volumes (i.e., 15-25 ml) of Purification Buffer.

4. Apply 0.5 ml of the hapten-carrier mixture directly to the center of the column disc. Add 8-10 aliquots of 0.5 ml of

Purification Buffer and collect each fraction in a separate tube.

5. Measure absorbance at 280 nm to locate fractions containing the conjugate. The hapten-carrier conjugate will be in the first absorbance peak detected. Pool all fractions that contain acceptable levels of conjugate.

6. After the conjugate-containing fractions have emerged, the non-conjugated hapten can be recovered by continuing to add

buffer to the column and collecting additional fractions.

7. If the immunogen is to be stored for more than a few days, sterile filter the conjugate fractions and store in a sterile

container at 4°C or -20C. See Appendix B for immunization suggestions.

Note: To purify antibodies specific to the peptide, prepare an affinity column by immobilizing the peptide through the same functional group used to prepare the immunogen (see Appendix C).

C. Checking

The conjugation can be checked by running the unconjugated BSA, and the AcBSA conjugated to the peptides. A band shift in the AcBSA lane is indicative of total conjugation.