The resonance energy transfer dye Cy7-Allophycocyanin (Cy7APC) is a
relatively new entrant onto the fluorescence field. It provides a bright third
color for dye laser (595 - 605 nm excitation) and a second color for HeNe
excitation (633 nm), with an emission at almost 800 nm. (See the fluorescence spectra). Cy7APC conjugates of
immunoglobulins are relatively straightforward to make, requiring an additional
step over the standard APC conjugation: the synthesis of the Cy7APC tandem dye.
Refer to the notes about resonance energy transfer dyes
for general information about the synthesis and testing of dyes like Cy7APC, as
well as the reference listed below for more detailed information.
Allophycocyanin can be purchased from several vendors, or isolated from the
algae directly (be prepared to spend 2+ weeks in the cold room).
Refer to notes about the following procedures used by this protocol:
Reductive cross-linking of antibodies
Column chromatography
Reagent storage
You can also use the short, less-detailed protocol for
reference.
I. Preparation of APC
II. Cy7 conjugation of APC
III. SMCC conjugation of APC
IV. Reduction of IgG
V. Covalent conjugation
Materials, Chemicals, and Buffers
Referencess
The entire conjugation can be performed in a single (long) 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 first time you make Cy7APC derivatives, you should probably make several
different conjugates with varying ratios of Cy7 to APC. Make 1-3 mg of each;
conjugate each to a test antibody. Compare the different conjugates for
brightness and compensation requirements; select the appropriate ratio of Cy7
to APC. In the protocol below, a molar ratio of 10 is suggested; this ratio
resulted in a dye with absorbance ratios measured at 755nm and 565nm of about
1.9. Such a tandem reagent has the good Cy7 fluorescence while not requiring
excessive compensation; however, molar ratios of 2 to 30 should be attempted in
initial trials. Once the appropriate ratio is selected, the reaction can be scaled
up to make the large enough quantities for many antibody conjugations.
Conjugate properties are determined by the absorbance ratio of the product,
which in turn, under consistent reaction conditions, should reflect input molar
ratios of the reactive dyes.
The SMCC-Cy7APC derivative (the result of step III) is quite stable (at least a
few months at 4C in the "Exchange Buffer"). As well, each different
lot of Cy7APC may have slightly different spectral characteristics (and thus
require different compensations). Therefore, it is best to derivatize 10-30 (or
more) milligrams of APC at the same time, and use it for several antibody
conjugations (even over a period of weeks). Steps IV and V 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-Cy7APC 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 and Cy7APC for each conjugation.
Dialyze or exchange the APC into "C Reaction 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 "C Reaction 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 APC react with the bis-Cy7 dye to yield a tandem
(resonance energy transfer) dye.
Dissolve the appropriate amount of bis-Cy7 in anhydrous DMSO to an effective
concentration of 10 mg/ml immediately prior to use. For a molar ratio of 10
(see notes above on using different ratios), add 91.1 nmol Cy7 per mg of APC.
Refer to the manufacturer's notes about the effective molecular weight of the
preparation of Cy7 you are using.
Incubate and rotate the foil-wrapped tube at room temperature for 60 minutes.
Purify the reaction mixture over a gel filtration column pre-equilibrated with
"Dialysis Buffer".
Take an absorbance spectrum of the Cy7APC conjugate to determine the degree of
Cy7 substitution as well as the concentration. (See an example absorbance spectrum for Cy7APC). A molar ratio of 10
Cy7 to 1 APC should result in a conjugate which has an A(755):A(655) ratio of
approximately 1.9. Significantly different ratios indicate that the reaction
proceeded too fast or too slow.
The amino groups on the Cy7APC 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 Cy7APC while vortexing. Wrap the reaction tube in
aluminum foil and rotate at room temperature for 60 minutes.
Pass the derivatized Cy7APC 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 Cy7APC (> 4
mg/ml) is desirable for such longer-term storage.
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 Cy7APC 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-Cy7APC per mg of IgG. Wrap the reaction tube in aluminum
foil and rotate for 60 minutes at room temp. Note: These molar ratios (~2 PE
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 Cy7APC 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.
Materials:
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), Amersham, catalog
No. 17-0851-01.
For <0.5 ml sample volumes: NAP5 columns (Sephadex G-25 DNA grade), Amersham,
catalog No 17-0853-02.
Chemicals:
Cy7 - Cy7-bis-OSU,
N,N'-biscarboxypentyl-5,5'-disulfonatoindotricarbocyanine
Amersham Life Science, catalog No. PA17000
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.
NaHCO3 - sodium bicarbonate
J. T. Baker, catalog No. 3508-05, mw 84.01
NaCO3 - sodium carbonate
J. T. Baker, catalog No. 3602-01, mw 106
NaCl - Sodium Chloride
Sigma, Catalog No S-3014, mw 58.44
TRIZMA pre-Set crystals 8.0 - Combination of Tris base and TrisHCl
Sigma, catalog No. T4753, average mw 141.8
NaN3 – Sodium Azide
Sigma, catalog No S-2002, mw 65
Buffers:
"C Reaction Buffer"
500 mM carbonate, pH 9.0
To make 1 Liter:
17g Na2CO3
28g NaHCO3
pH to9.0
Note: sodium azide cannot be added to this buffer
"Dialysis Buffer"
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
"Exchange Buffer"
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
"Storage Buffer"
10 mM Tris, 150 mM NaCl, 0.1% NaN3, pH 8.2
To make 1 Liter:
1.42g TRIZMA 8.0
8.77g NaCl
1g NaN3
pH to 8.2
See hints on storing buffers.
M Roederer, AB Kantor, DR Parks, and LA Herzenberg: Cy7PE and Cy7APC: Bright
new probes for immunofluorescence. Cytometry, 24:191-197 (1996).
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.