Texas Red (TR) is most commonly used on FACS machines with a dye laser
(excitation 595-605 nm). It emits at about 620 nm, much shorter than the APC
emission. (See the fluorescence spectra). It makes
for a relatively bright reagent, allowing for discrimination of
weakly-expressed antigens. Direct TR conjugates of antibodies, however, are
often sticky and are quite unstable, lasting only a few months at 4C.
Therefore, for many years TR was used only as an avidin conjugate, i.e., for
use as a second step for biotin-labeled antibodies. However, when TR is
conjugated to BSA as a carrier, it is no longer sticky nor unstable. A single
TR-BSA (typically carrying 3 TR molecules) is conjugated to an antibody in a
site-specific manner, resulting in a bright reagent that is stable at 4C for
extended periods of time and shows very little background binding.
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. Derivatization of TR-BSA
II. Reduction of IgG
III. Covalent conjugation
Materials, chemicals, and buffers
References
The entire conjugation can be performed easily in a single day. 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 measure protein concentration
spectrophotometrically.
The protocol is divided into three phases: derivitization of the TR-BSA,
reduction of the IgG, and coupling of the two. SMCC-derivitized TR-BSA can be
stored in the refrigerator for extended periods (probably many months). Thus,
this step can be undertaken once for several antibody conjugations subsequently
performed on separate days. However, considering the low cost of the TR-BSA,
this is probably not necessary.
The amino groups on the BSA react with the succinamide of SMCC to yield a
maleimide-labeled TR-BSA.
Dissolve the TR-BSA into "Dialysis Buffer", at a concentration of 10
mg/ml. Prepare 3 mg TR-BSA per mg of antibody.
Prepare a 10 mg/ml stock solution of SMCC in dry DMSO immediately prior to use.
Add 45 µl of SMCC per mg of TR-BSA while vortexing. Wrap the reaction tube in
aluminum foil and rotate at room temperature for 60 minutes.
Note: for conjugations which are poor or fail, it may help to increase or decrease
the molar ratio of SMCC to TR-BSA, or use an alternative heterobifunctional
crosslinking reagent.
Pass the SMCC-TR-BSA over a filtration column pre-equilibrated with
"Exchange Buffer". (See hints
on using columns with fluorescently-conjugated proteins). Calculate the
concentration of the SMCC-TR-BSA assuming a 90% recovery of starting material.
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 TR-BSA 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 3 mg of SMCC-TR-BSA per mg of IgG. Wrap the reaction tube in aluminum foil
and rotate for 60 minutes at room temp. Note: These molar ratios (~6 TR-BSA per
IgG) have worked very well; however, more BSA per IgG can only help ensure that
all of the MAb is conjugated. Unconjugated BSA will be washed away during cell
staining (or during protein A or G purification of the conjugate, if desired).
The cost of the TR-BSA (~$3.50/mg) is inconsequential compared to other
factors.
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 TR-BSA 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:
TR-BSA - Texas Red-conjugated Bovine Serum Albumin.
Molecular Probes, Catalog No. A-824 (3.2 mole TR per mole BSA)
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:
"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.
This protocol is based on an original protocol devised by Alan Stall, based
on the phycobiliprotein conjugation protocol by Randy Hardy.
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.