Allophycocyanin (APC) is emerging as one of the brightest fluorescent probes
for FACS analysis. APC is a large protein (approximate molecular weight 80 kd)
containing many fluors. Typically, only one APC molecule is conjugated to an
antibody. Nonetheless, by virtue of its huge absorption coefficienct and almost
perfect quantum efficiency, as well as emission in a region of the spectrum
with extremely low cellular autofluorescence, it is one of the brightest dyes
used today. It emits at about 680 nm, and can be excited by either a dye laser
(595-605 nm), or the cheaper HeNe laser (633 nm). (See the fluorescence spectra and the absorbance
spectrum). Direct APC conjugates are relatively easy to make.
Allophycocyanin can be purchased from several vendors, or isolated from the
algae directly (be prepared to spend 2+ weeks in the cold room).
There is a conjugation kit available from Prozyme, Inc., which uses essentially this protocol (although most of the steps have been done for you). The conjugation kit provides enough of all of the necessary materials to conjugate 1 mg of antibody; the entire procedure takes about 2 hours to complete (the kit eliminates steps I and II in the conjugation protocol below). If you are only performing a limited number of APC conjugations, this alternative is probably desirable.
Refer to notes about the following procedures used by this protocol:
Reductive cross-linking of antibodies
You can also use the short, less-detailed protocol for reference.
I. Preparation of APC
II. Derivitization of APC
III. Reduction of IgG
IV. Covalent conjugation
The entire conjugation can be performed in a single day. However, dialysis
of stored APC prior to conjugation can take 24-48 hours. In addition to the
materials listed below, you will need to have a solution of your antibody at a
concentration of at least 2 mg/ml. You should be familiar with how to use
desalting columns and how to take absorbance spectra.
The SMCC-APC derivative (the result of steps I and II) is quite stable (at least a few months at 4C in the "Exchange Buffer"). Therefore, it is best to derivatize 10 or more milligrams of APC at the same time, and use it for several antibody conjugations (even over a period of weeks). Steps III and IV together take only a few hours and minimal preparation; thus, storing the SMCC-derivative is very convenient. It is possible that long-term storage of the SMCC-APC may be best as a saturated ammonium sulfate precipitate--after which extensive dialysis similar to that in Step I below should be performed.
While taking absorbance spectra is not critical to the success of the procedure, it is highly recommended as a quality control, and as a permanent record of the quality of the APC for each conjugation.
Dialyze or exchange the APC into "Dialysis Buffer". Concentration
before derivatization is typically 5-10 mg/ml. Note: APC is most stable as a
SAS (sodium ammonium sulfate) precipitate prior to coupling. If the APC is
stored as a SAS precipitate, it must be extensively dialyzed prior to use.
Dialyze against 2 changes of 1 liter per ml APC of PBS before dialyzing against
1 liter per ml of "Dialysis Buffer".
Use 1.7 mg of APC per mg of IgG to be modified; this includes an extra 10% for loss during buffer exchanges.
To check the APC purity, measure the absorbance at 280, 620 and 655 nm. (1 mg/ml of APC has an OD at 655nm of 5.9). A 655/620 ratio >1.4 indicates adequate removal of contaminating phycocyanin; a 655/280 ratio > 4 indicates adequate removal of all other proteins. See the sample absorbance spectrum for APC.
The amino groups on the phycoerythrin (APC) react with the succinamide to
yield a maleimide-labeled APC.
Prepare a 10 mg/ml stock solution of SMCC in dry DMSO immediately prior to use.
Add 6 µl of SMCC per mg of APC while vortexing. Wrap the reaction tube in aluminum foil and rotate at room temperature for 60 minutes.
Pass the derivatized APC over a gel filtration column pre-equilibrated with "Exchange Buffer". See hints on using columns with fluorescent proteins. The SMCC-derivative is stable and may be stored at 4C for several weeks; a high concentration of APC (> 4 mg/ml) is desirable for such longer-term storage.
Note: for conjugations which fail or are poor, it may help to increase or decrease the amount of SMCC with respect to APC, or to use an alternative heterobifunctional crosslinking reagent.
The hinge disulfide bonds are reduced to yield free sulfhydryls.
Prepare a fresh solution of 1 M DTT (15.4 mg/100 µl) in distilled water.
IgG solutions should be at 4 mg/ml or higher for best results. The reduction can be carried out in almost any buffer; MES, phosphate, and TRIS buffers (pH range 6 to 8) have been used successfully. The antibody should be concentrated if less than 2 mg/ml. Include an extra 10% for losses on the buffer exchange column.
Make each IgG solution 20 mM in DTT: add 20 µl of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 ml fractions off the column; determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically (see hints on using columns for separation of nonfluorescent proteins).
Carry out the conjugation as soon as possible after this step.
Note: for conjugations which are poor or fail, it may help to reduce the DTT concentration.
The APC is covalently coupled to the IgG
through reaction of the maleimide groups with the free sulfhydryl on the IgG.
Do not delay this step since the IgG sulfhydryls will reoxidize.
Add 1.5 mg of SMCC-APC per mg of IgG. Wrap the reaction tube in aluminum foil and rotate for 60 minutes at room temp. Note: These molar ratios (~2 APC per IgG) have worked very well. For conjugations which fail or are poor, different molar ratios may help.
After 60 minutes, unreacted free sulfhydryls on the IgG must be blocked.
Prepare a fresh solution of 10 mg NEM in 1.0 ml dry DMSO.
Add 34 µg (3.4 µl) per mg of IgG. Wrap and rotate for 20 minutes at room temperature.
The product can be either dialyzed or exchanged over a column into an appropriate buffer (e.g. "Storage Buffer"). It is best to keep the product at high concentration (> 1 mg/ml) for optimal stability. Never freeze the congugates. It may be useful to spin APC conjugates prior to use in staining, especially if background seems to be a problem (e.g., at 10,000g in a microcentrifuge, at 4C). See also general hints on storing conjugates.
For column separations, we often use one of two types of pre-poured columns:
For 1.25ml to 2.5ml sample volumes: PD-10 (Sephadex G-25M), Pharmacia Biotech, catalog No. 17-0851-01.
For 0.5 to 1.5ml sample volumnes: KwikSep dextran desalting columns, Pierce, catalog No. 43232.
pHix - 5 mg/ml pentachlorophenol in 95% ethanol (use as 10,000x, or 3-4 drops per liter)
Sigma, catalog No. P1045
SMCC - succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
Pierce, catalog No. 22360, mw 334.42
NEM – N-Ethylmaleimide
Sigma, Catalog E-1271, mw 125.1
DMSO - anyhydrous dimethyl sulfoxide
Aldrich, catalog No. 27,685-5.
Note: keep the DMSO absolutely dry at all times. We keep the bottle in a dessicator. Pour out an amount of DMSO sufficient for your need and then pipette that; don't pipetter directly into the bottle.
DTT - Dithiothreitol
Sigma, catalog No. D-9779
Sodium Phosphate Dibasic (7*H2O)
J. T. Baker, catalog No. 3824-01, mw 268.07
EDTA - Ethylenediaminetetraacetic acid (Disodium salt: dihydrate)
Sigma, catalog No. E5134, mw 372.2
MES - (2-[N-morpholino]ethanesulfonic acid)
Sigma, catalog No. M3885
NaCl - Sodium Chloride
J. T. Baker, catalog No. 3624-01, mw 58.44
TRIZMA 8.0 - Combination of Tris base and TrisHCl
Sigma, catalog No. T4753, average mw 141.8
50 mM Sodium phosphate, 1 mM EDTA, pH 7.0
To make 1 Liter:
13.41g Sodium phosphate dibasic (7*H2O)
0.37 g EDTA
50 mM MES, 2 mM EDTA, pH. 6.0
To make 1 Liter:
9.76 g MES
0.74 gm EDTA
pH to 6.0
10 mM Tris, 150 mM NaCl, 0.1% NaN3, pH 8.2
To make 1 Liter:
1.42g TRIZMA 8.0
pH to 8.2
See hints on storing buffers.
This protocol is based on an original protocol devised by Randy Hardy, and
modified by Alan Stall and Aaron Kantor.
Hardy, RR: Purification and coupling of fluorescent proteins for use in flow cytometry. In: Handbook of Experimental Immunology, 4th ed. DM Weir, LA Herzenberg, C Blackwell, and LA Herzenberg, editors. Blackwell Scientific Publications, Boston, 1986, pp. 31.1-31.12.