Cascade Blue is a UV-excitable dye that can be used for immunofluorescence
labeling. When used with the 351/361 nm excitation lines of an Argon laser, it
is not very bright; usually only extremely high density antigens can be
well-resolved by Cascade Blue. However, when used with the 405 nm excitation
line of a Krypton laser, it becomes a useful dye with a brightness approaching
that of fluorescein. Emission is collected at 440 nm (see the fluorescence spectra).
Refer to notes about the following procedures used by this protocol:
Column chromatography
Reagent storage
You can also use the short, less-detailed protocol for
reference.
I. Preparation of antibody
II. Covalent conjugation
Materials, chemicals, and buffers
References
The entire conjugation can be performed in about a half-day. In addition to
the materials listed below, you will need to have a solution of your antibody
at a concentration (optimally) of at least 2 mg/ml. The extent of Cascade Blue
conjugation to the antibody may depend on the concentration of antibody in
solution; for consistent conjugations, use a consistent concentration. You
should be familiar with how to use a desalting column and how to take
absorbance spectra.
The reactive Cascade Blue molecule is unstable. Once the Cascade Blue is
solubilized, it should be used immediately.
When first conjugating an antibody, a range of Cascade Blue to antibody
concentrations should be compared. The protocol suggests 150 µg per mg of
antibody; for a first-time titration of Cascade Blue, try a range of 40 to 600
µg Cascade Blue per mg of antibody (for instance, 40, 80, 160, 320, and 600 µg
per mg). Compare each conjugate by staining (you should perform a titration of
antibody on cells for each reagent to determine the optimal staining
concentration). Select the conjugate with the brightest "positive"
cells which still has low background on "negative" cells.
Note: it is critical that sodium azide be completely removed from any
antibody.
Dialyze or exchange over a column the antibody in "B Reaction
Buffer".
Measure the antibody concentration after buffer equilibration. (For IgG, 1
mg/ml has an A(280) of 1.4). If the antibody concentration is less than 1
mg/ml, the conjugation will probably be sub-optimal. If necessary, dilute the
antibody to a concentration of 4 mg/ml.
Cascade Blue is covalently coupled to primary amines (lysines) of the
immunoglobulin.
Dissolve 5 mgs of Cascade Blue in 500 µl anhydrous DMSO immediately before use.
This is tedious and takes a bit of vortexing and time.
Add Cascade Blue to give a ratio of 150 µg per mg of antibody; mix immediately.
(See notes above about using different molar rations of Cascade Blue to
antibody).
Wrap the tube in foil; incubate and rotate at room temperature for 4 hours.
Remove the unreacted Cascade Blue and exchange the antibody into "Storage
Buffer" by gel filtration or dialysis. The unreacted dye will have the
apparent color on the column; usually, the antibody conjugate will be too low a
concentration to be colored: do not make the mistake of collecting the antibody
by color visualization! (You can use a hand-held UV lamp in a darkened room to
visualize the conjugate--it will appear faintly blue in comparison to the
buffer).
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:
Cascade Blue acetyl azide, trisodium salt
Molecular Probes catalog No. C-2284, mw 607.42
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.
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:
"B Reaction Buffer"
100 mM carbonate, pH 8.4
To make 1 Liter:
8.4g NaHCO3
pH to 8.4
Note: sodium azide cannot be added to this buffer
"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 Michael Anderson.