FluoroTag™FITC Conjugation Kit
Product Number FITC1
Storage Temperature 2−8 °C
TECHNICAL BULLETIN
Product Description
The FluoroTag FITC Conjugation Kit is suitable for the conjugation of polyclonal and monoclonal antibodies with fluorescein isothiocyanate (FITC) for u in immunohistochemistry and immunofluorescence studies utilizing flow cytometry. It may also be ud for conjugation of FITC to peptide hormones, cytokines, growth factors, and other proteins. The kit contains sufficient reagents for
at least 5 conjugations. Fluorescein isothiocyanate (FITC), Isomer I is among the most widely ud fluorescent labeling reagents due to the fluorophore’s high quantum efficiency and conjugate stability. FITC has an absorption maximum at 495 nm and emission maximum at 525 nm. FITC reacts with free amino groups of proteins to form stable conjugates (e Figure 1). FITC-protein conjugates, in particular FITC labeled antibodies, are ud as specific probes in immunocytochemistry and flow cytometry applications.1,2Biologically active FITC-conjugates of peptide hormones and growth factors have been successfully prepared which identify receptors on target cells.3,4FITC has also been ud as a site-specific probe for veral other proteins.5,6,7
The u of optimal labeling conditions is recommended. Overlabeling of proteins generally results in altered specificity, aggregation and/or precipitation of the protein. Fluorescent labeling of antibodies with high fluorophore to antibody ratios (molar F/P >6) usually results in incread non-specific binding (fluorescent background) and decread quantum yield due to the fluorophore lf-quenching effect.
The FluoroTag Kit includes detailed procedures for both small and large scale conjugation of FITC to antibody. Small scale FITC conjugations are performed using three different molar ratios of FITC to antibody. Bad on the molar ratio that gives the most satisfactory result, the large scale procedure
can then be performed to optimally label the protein. The labeled protein is purified from the unconjugated fluorescein by a quick Sephadex®G-25M column. The F/P molar ratio of the purified protein is then determined by measuring the absorbance at 280 nm and at 495 nm.
Reagents and Materials Provided
• Fluorescein isothiocyanate, Isomer I (FITC), F7250.
Five amber vials each containing 2.0 mg of
lyophilized fluorescein isothiocyanate.
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• 0.1M Sodium Carbonate-Bicarbonate Buffer, pH9.0, C0688. Five capsules containing powder.• Phosphate Buffered Saline (PBS), P3813. Five packages containing powder.
• Gel filtration columns. Two columns prepacked with Sephadex G-25M. The rve to parate
unreacted FITC from the conjugate and for buffer
拉斐尔简介exchange. The columns are preswollen in water
containing 0.15% Kathon®CG/ICP II as
prervative. The gel filtration columns are supplied for two different reaction scales:
四六级英语成绩查询入口Small Scale (Column A), B7533:One column
prepacked with Sephadex G-25M. The bed volume of the column is 3.5 ml and the bed height is
2.6cm. The maximal sample volume is 0.3 ml.
Large Scale (Column B), B4783:One column
prepacked with Sephadex G-25M. The bed volume of the column is 9.1 ml and the bed height is 5cm.
The maximal sample volume is 1.5 ml.
Reagents and Equipment Required but Not Provided
• Standard glass vials (1.5−2 ml capacity) equipped with stirring bars.
• Two dilution vials are needed for each conjugation.• Vortex mixer.
• Standard glass tubes (12 X75 mm or 13 X100 mm) to collect fractions from Sephadex G-25M columns.• Aluminum foil -To protect reaction and FITC labeled protein from inten light.
• Quartz cuvette -1 cm path length
• UV/Visible spectrophotometer.
2
Precautions and Disclaimer
This product is for R&D u only, not for drug, houhold, or other us. Plea consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.
Preparation Instructions
0.1 M Sodium Carbonate-Bicarbonate Buffer, pH 9.0,
C0688-Add contents of one capsule to 50 ml of deionized water to make 0.1M sodium carbonate-bicarbonate buffer, pH9.0. This is ud to dissolve FITC and to buffer the conjugation reaction. Phos
igiphate Buffered Saline (PBS), P3813-Mix contents of one package with 800 ml of deionized water. Adjust volume to 1 liter to make 10 mM sodium phosphate buffer, 27 mM KCl, 138 mM NaCl, pH 7.4. This rves as an equilibration buffer for the Sephadex G-25 columns, for the elution of the labeled protein from the column, and for the final dilution of the labeled protein.
Storage/Stability
Store at 2−8 °C.
Procedure
The protocol outlined describes the labeling of 1 mg of IgG at 5 mg/ml with FITC (Small Scale Conjugation Procedure). The procedure can be scaled up to 5 mg of IgG maintaining the same concentration and molar ratio of the reagents (Large Scale Conjugation Procedure). It is important to consider that the number and surface availability of amine groups (primarily -amine groups of lysine residues) vary greatly among proteins and even among different IgGs. This may result in a large variability of the level of labeling. Testing different FITC to antibody molar ratios to determine the optimal levels of labeling of the antibody is recommended.
Small Scale Conjugation Procedure(1.0 mg IgG)
This procedure describes the conjugation of FITC to
1mg of IgG, using one of the following molar ratios in the reaction mixture: 5:1, 10:1 and 20:1 of FITC
(MW389) to IgG (MW 150,000). The labeling is performed in a final reaction volume of 0.25 ml. In general, the reaction molar ratios result in fluorescein-antibody conjugates with F/P ratios of 1−2, 2−4 and 3−6, respectively. This procedure can be modified if a protein of a different molecular weight (or a different amount of IgG) is ud in the labeling reaction. (See Table 2 reaction mixture ratios for whole IgG, IgM, or antibody fragments.)1.Dissolve the contents of one sodium carbonate-
bicarbonate capsule (C0688) in 50 ml of deionized water. The pH of this buffer should be 9.0 ±0.1
(See Application Notes 1&2).
2.Prepare at least 0.25 ml of antibody solution at
5.0mg/ml in 0.1 M carbonate-bicarbonate buffer,
pH 9.0 (See Application Notes 3, 4 & 5). The A280
of an IgG solution at 1.0 mg/ml is 1.4 (1.0 cm path length).
3.Add 0.2 ml (1.0 mg) of the antibody solution into
each reaction vial labeled “5:1”, “10:1”, or “20:1”. 4.Reconstitute one vial of FITC (F7250) in 2 ml of
0.1M carbonate-bicarbonate buffer and vortex until
all FITC has dissolved. Label vial “20:1 FITC”. The solution should be freshly prepared before each
conjugation and ud within 5 minutes (See
Application Note 6). The "20:1 FITC" solution is
further ud to prepare the "5:1" and "10:1"
solutions.
5.Prepare the required dilution of FITC in 0.1M
carbonate-bicarbonate buffer as directed in
Table1.
Table 1.
Dilution of FITC in 0.1M Carbonate-Bicarbonate Buffer
6.Add 50 µl of the appropriate FITC dilution dropwi
while stirring to the correspondingly labeled
reaction vial.
7.Completely cover the reaction vial with aluminum
foil to protect from light.
8.Incubate all reaction vials for 2 hours at room
temperature with gentle stirring.
Isolation of labeled protein
1.Empty contents of PBS package (P3813) into a
suitable container. Add 800 ml of distilled or
apple said yesdeionized water and mix. Adjust to final volume of 1,000 ml.
2.Label Sephadex G-25M, column A (B7533), “5:1”,
“10:1”, or “20:1”. Support column over a suitable
(100ml) beaker.
3.Remove cap from the top of the column, cut open
lower tip of column and let excess of liquid flow
through. The column will not run dry.
3
4.Equilibrate the column with 12 ml of PBS solution
(6 X2 ml). If the column is not immediately ud,
clo with top and bottom caps and store at 2−8 °C.
5.Apply reaction mixture to top of column gel bed and
collect the flow through (Fraction 1)
6.Elute the column with 2.5 ml of PBS, collecting
0.25ml fractions (10 X0.25 ml). Monitor the
absorbance of each fraction at 280 nm. Two bands will be visible during elution. The conjugate is
prent in the first band (fractions 6−8)
(See Application Note 7).
7.Pool the main fractions. Do not collect fractions with
A280<0.2.
8.Wash the column with 35 ml (10 X column volumes)
of PBS to remove unbound fluorophore. This is
sufficient to regenerate the column.
9.For prolonged storage, wash the column with 10ml
of PBS containing 0.05% sodium azide and store
capped at 2−8 °C, with 1 ml buffer above the gel. 10.Determine the fluorescein/protein ratio (F/P) of the
conjugate using a spectrophotometer as described in Determination of Fluorescein/Protein Molar Ratio (F/P).
11.For storage of the conjugate after determination of
the F/P molar ratio, add 1% (w/v) BSA and 0.1%
(w/v) sodium azide to the conjugate. Store at
2−8°C, protected from light.
Large Scale Conjugation Procedure(5.0 mg IgG)
This procedure describes the scale-up of the conjugation reaction of FITC to 5 mg of IgG, using the molar ratio, which gives the most satisfactory results as obtained from the “Small Scale Conjugation Procedure.” The labeling is performed maintaining the same concentrations and ratios of reagents ud previously. The reaction is performed in a final reaction volume of 1.25 ml.
1.Add 1.0 ml (5.0 mg) of the antibody solution to a
reaction vial labeled “5:1”, “10:1”, or “20:1”.
2.Reconstitute one vial of FITC (F7250) in 2 ml of
0.1 M carbonate-bicarbonate buffer and vortex until
all FITC has dissolved.
3.Prepare 10:1 or 5:1 dilution of FITC in 0.1M
carbonate-bicarbonate buffer as directed in
Table1, if necessary.
4.Add 250 µl of the appropriate FITC dilution
dropwi while stirring to the reaction vial.
5.Completely cover the reaction vial with aluminum
foil to protect from light.
6.Incubate reaction vial for 2hours at room
temperature with gentle stirring.Isolation of labeled protein
1.Label Sephadex G-25M, column B (B4783), “5:1”,
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“10:1”, or “20:1”. Support the column over a
suitable (100 ml) beaker.
2.Remove cap from the top of the column, cut open
lower tip of column and let excess of liquid flow
through. The column will not run dry.
3.Equilibrate the column with 30 ml of PBS solution
(6 X5 ml). If the column is not immediately ud,
clo with top and bottom caps and store at 2−8 °C.
4.Apply reaction mixture to top of the column gel bed
and collect flow through (Fraction 1).
5.Elute column with 10 ml of PBS, collecting 1.0 ml
fractions (10 X1 ml). Monitor the absorbance of
each fraction at 280 nm. Two bands will be visible during elution. The conjugate is prent in the first band (fractions 3−5). (See Application Note 7.)
6.Pool the main fractions. Do not collect fractions
with A280<0.4.
7.Wash the column with 50 ml of PBS solution to
remove unbound fluorophore. This is sufficient to
regenerate the column.
8.Prerve the column as instructed in step 9 of the
small scale “Isolation of Labeled Protein” ction. Determination of Fluorescein/Protein Molar Ratio (F/P) The F/P molar ratio is defined as the ratio of moles of FITC to moles of protein in the conjugate.
To determine this ratio, it is necessary to first determine the absorbance of the conjugate sample at 280 nm and then at 495 nm.
1.Place the conjugate sample in a quartz cuvette.
For the large scale conjugation, dilute 0.1 ml of the FITC conjugate in 0.9 ml of PBS containing sodium azide. Read the absorbance of the conjugate
sample at 280 nm and 495 nm. The absorbance
reading of the conjugate sample should be between
0.2 and 1.4 at 280 nm. If the absorbance reading is
outside this range, adjust the sample dilution
accordingly.
2.From the absorbance readings (A280and A495) of
the conjugate sample, calculate the F/P of the
fluorescein-IgG conjugate according to the
8
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The protein concentration of the fluorescein-IgG
傻瓜国际音标下载
FITC, u the general formula below, substituting the appropriate values for the particular protein:
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MW is the molecular weight of the protein.389 is the molecular weight of FITC.195 is the absorption E 0.1% of bound FITC at 490 nm at pH 13.0.(0.35 x A 495) is the correction factor due to the absorbance of 280
(For E 0.1%and C values, e Table 2)FITC at 280 nm.8E 0.1%is the absorption at 280 nm of a protein at 1.0mg/ml.280280
Application Notes
1.Do not store sodium carbonate-bicarbonate buffer
more than 1 week at 2−8 °C. The pH of the buffer may change upon storage. It is advid that fresh buffer be made just before u.
2.The conjugation of FITC to proteins is affected by
the reaction conditions (concentration, temperature, pH).
叟猴3.When conjugating antibodies with FITC, the starting
material should be free of contaminating rum proteins. Affinity isolated antibodies, IgG fractions, or Protein A purified immunoglobulin are generally acceptable.
4.Protein solutions should not be prepared in buffers
containing amines such as Tris, glycine or sodium azide since they inhibit the labeling reaction. If the buffer contains amines or sodium azide, dialyze protein solution (1 ml) against PBS, pH 7.4
(1,000ml), overnight at 2−8 °C. Avoid dialysis at high pH values (>8.0−8.5) as this may be harmful to some proteins.
5.If the antibody is in PBS pH 7.4 (without azide), add
1 M carbonate-bicarbonate buffer (1 capsule (C0688) in 5 ml deionized water) to a final concentration of 0.1 M (e.g., 0.1 ml of 1 M
carbonate-bicarbonate buffer to 0.9 ml IgG solution at 5.0 mg/ml).
6.FITC is not stable in aqueous solutions. Do not
store FITC stock solutions.
7.The cond band containing unbound fluorophore
is retained on the column and will elute only with subquent PBS washes.References
1.Staines, W., et al., Three-color
immunofluorescence histochemistry allowing triple labeling within a single ction. J. Histochem. Cytochem., 36, 145 (1988).
2.Carter, N., in Flow Cytometry: A Practical
好的英语学习网站Approach, M.G. Ormerod, Ed., IRL Press (1990).3.Carraway, K., et al., Location of the epidermal
growth factor binding site on the EGF receptor. A resonance energy transfer study. Biochemistry , 29, 8741 (1990).
4.Schwartz, J., and Vale, W., Fluorescent and
cytotoxic analogs of corticotropin-releasing factor: probes for studying target cells in heterogeneous populations. Methods in Enzymol ., 168, 29 (1989).5.Adams, S., et al., Fluorescence ratio imaging of
cyclic AMP in single cells. Nature , 349, 694 (1991).6.Abbott, A., et al., Immunochemical and
spectroscopic characterization of two fluorescein 5'-isothiocyanate labeling sites on Na+, K(+)-ATPa. Biochemistry , 30, 1692 (1991).
7.Pavalko, F., and Burridge, K., Disruption of the
actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin. J. Cell Biol.,114, 481 (1991).
8.The, T., and Feltkamp, T., Conjugation of
fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunol.,18, 865 (1970).Sephadex is a registered trademark of Pharmacia, Inc. Kathon is a registered trademark of the Rohm & Haas Company.
LPG,KTA 12/05-1
5 Table 2.
Reaction Mixture Ratios for Whole IgG, IgM or Antibody Fragments
** This corresponds to volume in microliters of the appropriate FITC concentration (5:1, 10:1, or 20:1).
***For IgM, a lower molar ratio is recommended as high levels of FITC may result in overlabeling of the antibody and lf-quenching of the fluorophore.
****For F(ab′) 2 u a 2-fold more concentrated FITC solution than ud for IgG.
*****For Fab′and Fc u a 4-fold more concentrated FITC solution than ud for IgG.
