PE-Protein Conjugation

Below is a brief protocol detailing PE-protein conjugation utilizing SMCC and DTT. For an alternative protein conjugation technique, see our Buccutite PE Kit.

Note: This protocol uses IgG (MW: 150 kDa) as its protein for conjugation, though other proteins may be substituted with appropriately modified amounts.

PE Preparation

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Notes: PE must first be centrifuged if it is stored as a solution (generally as an ammonium sulfate). Centrifuge and discard the supernant, and then reconstitute the precipitate in PBS at the same concentration as the original solution.

If the PE is stored as a solid, suspend immediately in PBS.

Use 3.5 mg of PE for each mg of IgG you plan to be conjugated.

1. Dialyze the PE solution against 1 liter of PBS per mL of PE. Repeat once.
2. Dialyze the PE solution against 1 liter of Dialysis Buffer* per mL of PE.
3. Ensure the purity of your PE solution by measuring its absorbance at 280, 565, and 620 nm. A resulting ratio of 565/620 nm readings greater than 50 is an indicator of the removal of phycocyanin, and a 565/280 ratio greater than 5 indicates further purity of the solution.

PE Derivatization (SMCC-PE)

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1. Immediately prior to its use in the following steps and conjugation, prepare a 10 mg/mL stock of SMCC in DMSO.
2. Add 11µl of the SMCC solution per mg of PE to the PE solution created from step 1b. Wrap the solution in aluminum and let incubate for 1 hour, rotating.
3. Equilibriate a gel filtration column with Exchange Buffer*, and pass the derivatized PE from the previous step over it.

Reduce the IgG

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1. Prepare a solution of 1M DTT (15.4 mg/100µl) in distilled water.
2. Prepare your IgG in a suitable buffer at 4mg/mL or higher.
3. Create a 20mM IgG solution by adding 20µl of DTT stock per mL of IgG solution while mixing. Let stand for 30 with no additional mixing.
4. Equilibriate a filtration column with Exchange Buffer, and pass the reduced IgG over it. Collect 0.25 mL fractions from the column and pool with majority.

Carry out the following conjugation as soon as this step, and step 2 are completed (simultaneously).

Conjugation

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1. Add 3.2 mg of SMCC-PE per mg of the reduced IgG solution. Wrap in aluminum and rotate for 1 hour at room temperature.
2. Prepare a solution of 10 mg NEM in 1 mL of DMSO.
3. Add 34µg to the IgG solution per mg of IgG. Wrap in aluminum again and rotate at room temperature for 20 minutes.
4. Final solution can be dialyzed or run over a column into a suitable buffer.

Note: Do not freeze conjugates.