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Phycobiliproteins Frequently Asked Question (FAQ)

We would not suggest using spin desalting columns, as they don’t have the resolution to completely remove the ammonium ions.  We would suggest using one of the following options for desalting:

1.  Dialyze the APC in PBS or your buffer of choice (be sure to perform several buffer changes over at least a day to ensure complete exchange of the ammonium ions).  Standard dialysis tubing or commercial dialysis cassettes or cartridges can be used, intact APC is around 100 kDa.

2.  Desalt using a column such as a PD-10 column from GE.  In this case the APC should be pelleted by centrifugation (and the colorless supernatant discarded) and the pellet completely redissolved in PBS (or your buffer of choice)  prior to loading on the column. This can also be used to jump-start the dialysis option above if time is a factor. We suggest a sample volume that is no more than 10% of the column bed volume, to be safe.

We don’t have specific protocols for the use of PB11 for instrument calibration, but Turner Designs should be able to assist. However, we do have some handling recommendations.

We would recommend taking only what you need from the vial and storing the rest undiluted at 4°C in the dark, in the original ammonium sulfate formulation buffer. While PB11 can be diluted in water, it does not store well this way.

PB11 is not very stable when diluted in water or PBS, so only the amount needed for a single calibration should be diluted at one time and each calibration should be done with a fresh dilution of PB11. Because the dilutions will be made from fresh material, it is acceptable to use water. Since the concentration of PB11 will be very small, it is not necessary to dialyze the material to remove the ammonium sulfate prior to dilution.

One important consideration is that PB11 must be thoroughly mixed before pipetting any material out of the original container for dilution.  Our concentration specification for PB11 is ≥10 mg/ml, but often we supply the material at a higher concentration. The concentration is listed on the Certificate of Analysis.

PB40 PerCP can be used for conjugation to protein, but will have to be activated with SMCC. Buffer exchange the PerCP into PBS, and activate with NHS-SMCC (ratios may have to be optimized).

We advise you should only activate as much material as you need for a conjugation, since the shelf life of activated PerCP is short. For this reason we do not offer a PerCP conjugation kit, or activated PerCP as a standalone product as we do for RPE and APC. A reasonable starting point for an antibody conjugation would be 1 mg SMCC-activated PerCP:1 mg Ab, but this will need to be optimized for your particular target.
For general conjugation protocols, our PJ31K RPE Conjugation Kit describes the procedure and reagents used.
Some monoclonal antibodies may precipitate upon DTT reduction used in e.g. PJ31K protocol. An alternative protocol using SPDP/TCEP may be appropriate for antibodies that don’t fare well in DTT, and is provided in our Alternative Conjugation Protocols Tech Note.

PB32 is produced from the same Porphyra-like algal strain as PB31, but undergoes an additional purification step to remove free RPE subunits. We believe PB32 is a suitable replacement for PB31, and may provide better results in conjugation applications. However, if you would prefer to use PB31, please contact us.

We measure RPE concentration at 566 nm. Multiply the absorbance at λmax (566 nm) by 10 and divide by the extinction coefficient (82) to yield the concentration of the RPE (mg/ml).

RPE concentration (mg/ml) = (A566 measurement x 10) / 82

We give our extinction coefficients as E1% at λmax, which is the absorbance of a 1% solution (10 mg/ml) in a cuvette with a 1 cm path length. However, many scientists are used to seeing extinction coefficients for proteins at 1 mg/ml (0.1%). For RPE this works out as an A566 absorbance value of 8.2 for a 1 mg/ml solution, and makes for a simpler calculation:

RPE concentration (mg/ml) = A566 measurement / 8.2

We measure concentration spectrophotometrically rather than fluorometrically. Further information on calculating phycobiliprotein concentration & molarity is available in our Conjugate Evaluation tech note.

We would not suggest using spin desalting columns, as they don’t have the resolution to completely remove the ammonium ions.  We would suggest using one of the following options for desalting:

1.  Dialyze the RPE in PBS or your buffer of choice (be sure to perform several buffer changes over at least a day to ensure complete exchange of the ammonium ions).  Standard dialysis tubing or commercial dialysis cassettes or cartridges can be used, intact RPE is around 240 kDa.

2.  Desalt using a column such as a PD-10 column from GE.  In this case the RPE should be pelleted by centrifugation (and the colorless supernatant discarded) and the pellet completely redissolved in PBS (or your buffer of choice)  prior to loading on the column. This can also be used to jump-start the dialysis option above if time is a factor. We suggest a sample volume that is no more than 10% of the column bed volume, to be safe.