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), Pharmacia Biotech,
catalog No. 17-0851-01.
For 0.5 to 1.5ml sample volumnes: KwikSep dextran desalting columns, Pierce,
catalog No. 43232.
Chemicals:
TR-BSA - Texas Red-conjugated Bovine Serum Albumin.
Molecular Probes, Catalog No. A-824 (3.2 mole TR per mole BSA)
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. 22320, mw 334.42
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
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:
10.90 g MES
0.74 gm EDTA
pH to 6.0
"Storage Buffer"
10 mM Tris, 150 mM NaCl, pHix, pH 8.2
To make 1 Liter:
1.42g TRIZMA 8.0
8.77g NaCl
3-4 drops pHix
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.