 |
 |
 |
 |
from: h1913szo@ella.hu (Janos Szollosi 12/16/92)
to: cytometry@flowcyt.cyto.purdue.edu
Subject: TRITC labeling
Here I send my FITC and TRITC labeling protocol. I have used
this protocol for more than 7 years with very good results. I
hope that other persons in the cytnet will also use it with
success.
Janos Szollosi
Department of Biophysics,
Medical University School of Medicine,
Nagyerdei krt. 98
H-4012 Debrecen, Hungary
Telephone: (36) (52) 12-623
FAX: (36) (52) 12-623
E-mail: h1913szo@ella.hu
Labeling of IgG with FITC and TRITC.
References:
For labeling:
1. DePetris, S. (1978) Methods in Membrane Biology, Vol: 9, (Ed:
Korn, E.D.) Plenum Press, New York, p. 1-201.
For TRITC purification:
2. Spack, E.G.Jr., Packard, B., Wier, M.L., Edidin, M. (1986)
Hydrophobic adsorption chromatography to reduce nonspecific
staining by rhodamine-labeled antibodies. Anal. Biochem. 158:233
Brief Protocol:
Chemicals:
FITC - Molecular Probes, Polyscience, Isomer 5.
TRITC - Molecular Probes, Isomer R. (Open fresh vial for each
labeling!)
IgG - Purified antibodies, make sure that the solution does not
contain carrier proteins such as BSA and gelatin. The
concentration of the protein should be between 1 mg/ml and 15
mg/ml, and the smallest amount we can label is between 0.5 and
1 mg protein total. (In case of the TRITC labeling the protein
concentration should not be higher than 5-7 mg/ml because of the
lower solubility of the TRITC in the buffer solution.
Precipitation can be observed at high TRITC and protein
concentration.)
Solutions:
IgG solution
Set the pH to 9.3, via addition of Na2CO3 (0.5 M) solution, or
dialysis (overnight at 4 oC) against 0.1 M Na2CO3 (pH 9.3) or 0.1
Na3BO3 (pH 9.3). (If there is precipitation, remove it by
centrifugation, 3000 g, 5 min.)
Protein concentration - Determine using optical density
measurements at 280 nm. OD280,p is 1.4 for IgG solution of 1
mg/ml. Molar concentration can be calculated using MW = 160,000
for IgG.
FITC solution
Dissolve 1 or 2 mg FITC in 500 ul carbonate of borate buffer.
Spin it down (3000 g, 5 min), discard the pellet. Make 2000
times dilution, 50x40 in PBS or in borate buffer. Use the FITC
stock solution within one or two hours.
FITC concentration - Determine using the extinction coefficients
of FITC at 495 nm. Epsilon495,FITC = 75,000 at pH 9.3 and 68,000 at
pH 7.2. Measure the OD495/OD280 ratio (rFITC) as well as we need it
for the determination of the labeling ratio. This value should
be between 3.0 and 3.3. MWFITC = 389.
TRITC solution
Dissolve 1 or 2 mg TRITC in 300 ul DMSO. (You can try to
dissolve the TRITC in borate buffer, but the solubility of the
TRITC is not so good.) Spin down (3000 g, 5 min), discard the
pellet. Make 1000 times dilution, 20x50 in borate buffer. Use
the TRITC stock solution within one or two hours.
TRITC concentration - Determine using the extinction
coefficients of TRITC at 553 nm. Epsilon553,TRITC = 35,000 at pH
9.3. Measure the OD553/OD280 ratio (rTRITC) as well as we need it
for the determination of the labeling ratio. This value should
be between 0.80 and 3.0. (There is a great variability in this
value among different batches of TRITC. According to my
experience the higher is the value the better. In addition check
the absorption at 514 nm, if this shoulder is equal or exceeds
tha absorption peak at 553 nm, do not use this batch for
labeling.) MWTRITC = 479.
Labeling Procedure:
For FITC (and TRITC)
Add 15-20 ug FITC (30-40 ug/ml TRITC) to each mg protein. If the
protein concentration is below 5 mg/ml, the FITC (TRITC)
concentration should not be lower than 100 ug/ml (200 ug/ml).
(When relabeling the same kind of protein, you can reconsider the
concentrations according to the result of previous labeling,
since there is some variability in the resulting labeling ratio,
depending on the IgG type.) Incubate at room temperature for half
an hour.
Purification:
On Sephadex G25 column equilibrated with PBS. Size of the column
is 1x10 cm for 1 ml solution. (For FITC labeling shorter column
is also satisfactory.) After this step the FITC conjugated
proteins are ready to use. (The separation of free and bound dye
is really good for the FITC but not so good for the TRITC.)
In case of TRITC labeling additional steps are necessary to
remove the noncovalently bound, sticky TRITC molecules from the
IgG. Leave the partially purified (after Sephadex G25) TRITC-IgG
solutions in refrigerator for 2-4 days. There will be some
precipitation during this time period. Remove the precipitation
by centrifugation (3000 g, 5 min). SM-2 beads should be applied
to purify the TRITC-IgG. SM-2 beads should be treated as
described in the BioRad catalog, washed 5X with methanol, stored
in methanol. Before usage change the methanol to water, then to
PBS. Purification can be performed in two ways:
Batch technique - add 50 ul beads to 500 ul TRITC-IgG solution.
Incubate overnight. Remove the beads. If the beads are pink
repeat the treatment. Two or three treatments should be
satisfactory.
Column technique - Prepare a column 1x5 cm. Apply the TRITC-IgG.
The flowrate should be slow! The flowthrough is the purified
TRITC-IgG.
Determination of the labeling ratio:
For FITC-IgG:
[IgG] mg/ml = (OD280,FITC-IgG - OD495,FITC-IgG/rFITC)/1.4
[IgG] M = ([IgG] mg/ml)/160,000
[FITC] M = (OD495,FITC-IgG)/63,000
(There is some quenching int the FITC absorption due to the
conjugation)
F/P = ([FITC] M)/([IgG] M)
The F/P value should be between 2 and 4. 1 and 5 is O.K. but not
the best.
For TRITC-IgG:
[IgG] mg/ml = (OD280,TRITC-IgG - OD553,TRITC-IgG/rTRITC)/1.4
[IgG] M = ([IgG] mg/ml)/160,000
[TRITC] M = (OD553,TRITC-IgG)/35,000
R/P = ([TRITC] M)/([IgG] M)
The R/P values should be between 2-5. Because of the great
variability and uncertainty in the rTRITC value the R/P values are
not as accurate as the F/P values. The protein concentration can
be determined more accurately if competition with non-labeled IgG
or other methods (independent of TRITC absorption) are used.
Checking of TRITC-IgG:
1. Positive cells labeled with TRITC-IgG should have ring-like
fluorescence under the fluorescence microscope.
2. The fluorescence intensity of cells checked by FACS should
reach a saturation value with increasing TRITC-IgG concentration.
3. TRITC-IgG should compete with non-labeled IgG.
4. Non-relevant, negative cells should show no fluorescence if
they are labeled with TRITC-IgG.
Storage:
1. In the presence of 0.1% sodium azide the conjugated IgG
solutions can be kept in refrigerator for a year.
2. For long storage (or in the absence if sodium azide after a
month) fluorescently labeled IgG should be frozen in small
aliquots (50 to 100 ul). Avoid unnecessary freezing and thawing!
Make sure that the storage tubes are sealed well!
|
|
|
|