Methods in Molecular Biology
TM
HUMANA PRESS
Glycoprotein
Methods
and Protocols
Edited by
Anthony P. Corfield
VOLUME 125
The Mucins
Methods in Molecular Biology
TM
HUMANA PRESS
Edited by
Anthony P. Corfield
The Mucins
Glycoprotein
Methods
and Protocols
Isolation of Large Gel-Forming Mucins 3
3
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
1
Isolation of Large Gel-Forming Mucins
Julia R. Davies and Ingemar Carlstedt
1. Introduction
The large gel-forming mucins, which form the major macromolecular components
of mucous secretions, are members of the mucin “superfamily.” Nine mucin genes
(MUC1–MUC4, MUC5AC, MUC5B, and MUC6–MUC8) have been identified (for
reviews see refs. 1 and 2), with each gene showing expression in several tissues. Only
the MUC1, MUC2, MUC4, MUC5, and MUC7 mucins have been sequenced com-
pletely (3–11) although large stretches of MUC5AC (12–15) as well as the C-terminal
sequences of MUC3 (16) and MUC6 (17) are now known.
A characteristic feature of mucins is the presence of one or more domains rich in
serine and/or threonine residues that, owing to a high degree of oligosaccharide substi-
tution, are resistant to proteolysis. Mucins comprise cell-associated, usually mono-
meric species, as well as those that are secreted; the latter can be subdivided into large,
gel-forming glycoproteins and smaller, monomeric ones. The gel-forming mucins
(M
r
= 10–30 million Dalton) are oligomers formed by subunits (monomers) joined via
disulfide bonds (for a review see ref. 18), and treatment with reducing agents will
release the subunits and cause unfolding of regions stabilized by intramolecular disul-
fide bonds. Thus, after reduction, we term the monomers reduced subunits. Reduced
subunits are more sensitive to protease digestion than the intact mucin molecules.
The isolation procedures that we use for the large oligomeric mucins depend on
their source. In secretions such as respiratory tract sputum, tracheal lavage fluid, and
saliva, the material is centrifuged to separate the gel from the sol phase, allowing the
identification of the gel-forming mucins. Repeated extraction of the gel phase solubi-
lizes the “soluble” gel-forming species, leaving the “insoluble” mucin complex in the
extraction residue. Mucin subunits may be isolated from the “insoluble” glycoprotein
complex following reduction of disulfide bonds. When mucins are isolated from tissue
samples, it may be an advantage to “physically” separate histologically defined areas
of the tissue such as the surface and the submucosa of an epithelium. For example,
material from the surface epithelium may be enriched by gently scraping the surface
mucosa, thereby allowing gland material to be obtained from the remaining tissue.
4 Davies and Carlstedt
To isolate mucins, the bonds that hold the mucous gel together and those that anchor
cell-associated glycoproteins to the plasma membrane must be broken. In our labora-
tory, high concentrations of guanidinium chloride are used for this purpose, and high-
shear extraction procedures are avoided to minimize the risk of mechanical degradation.
Protease inhibitors are used to protect the protein core and a thiol blocking agent is
added to prevent thiol-disulfide bond exchange. However, breaking intermolecular
bonds with highly denaturing solvents will most likely cause unfolding of ordered
regions within the mucins, and properties dependent on an intact protein core structure
may be lost. Following extraction, mucins are subjected to isopycnic density gradient
centrifugation in the presence of guanidinium chloride. This method allows the group
separation of large amounts of mucins from nucleic acids and proteins/lipids under
dissociative conditions without the problems associated with matrix-based methods
such as gel chromatography.
2. Materials
2.1. Extraction of Mucins
2.1.1. Guanidinium Chloride Stock Solution
We use practical grade guanidinium chloride that is treated with activated charcoal
and subjected to ultrafiltration before use. We request small samples from several
companies and test them for clarity after filtration as well as absorbance at 280 nm.
Once we have established a suitable source, we purchase large batches of guanidinium
chloride, which considerably reduces the cost. Ultrapure grade guanidinium chloride,
which is much more expensive, may be used without prior purification.
1. Dissolve 765 g of guanidinium chloride in 1 L of distilled water, stirring constantly.
2. Add 10 g of activated charcoal and stir overnight.
3. Filter solution through double filter paper to remove the bulk of the charcoal.
4. To remove the remaining charcoal, filter solution through an Amicon PM10 filter
(Amicon, Beverley, MA), or equivalent, using an ultrafiltration cell. A Diaflow system is
a practical way to increase the filtration capacity.
5. Measure the density of the solution by weighing a known volume in a calibrated pipet,
and calculate the molarity of the guanidinium chloride stock solution (see Note 1). The
molarity should be approx 7.5 M with this procedure.
2.1.2. Solutions for Mucin Extractions
1. 6 M Guanidinium chloride extraction buffer: 6 M guanidinium chloride, 5 mM
EDTA, 10 mM sodium phosphate buffer, pH 6.5 (adjusted with NaOH). This solution
can be stored at room temperature. Before extraction, cool to 4°C and immediately
before use, add N-ethyl maleimide (NEM) and diisopropyl phosphofluoridate (DFP)
to final concentrations of 5 and 1 mM, respectively. DFP is extremely toxic (see
Note 2).
2. Phosphate buffered saline (PBS) containing protease inhibitors: 0.2 M sodium
chloride, 10 mM EDTA, 10 mM NEM, 2 mM DFP, 10 mM sodium phosphate buffer,
pH 7.4 (adjusted with NaOH).
Isolation of Large Gel-Forming Mucins 5
3. 6 M Guanidinium chloride reduction buffer: 6 M guanidinium chloride, 5 mM
EDTA, 0.1 M Tris/HCl buffer, pH 8.0 (adjusted with HCl). This solution can be stored
at room temperature.
2.2. Isopycnic Density Gradient Centrifugation
Density gradient centrifugation in our laboratory is carried out using CsCl in a
two-step procedure (see Notes 3 and 4).
1. Small samples of high-quality CsCl are obtained from several companies and tested for
clarity in solution, absorbance at 280 nm, and spurious color reactions with the analyses
for, e.g., carbohydrate that we use. Once we have established a suitable source, we pur-
chase large batches, which considerably reduces the cost. As with guanidinium chloride,
more expensive ultrapure grade may also be used.
2. Beckman Quick Seal polyallomer centrifuge tubes (Beckman Instruments, Palo Alto, CA)
or equivalent.
3. 6 M Guanidinium chloride extraction buffer, pH 6.5 (see Subheading 2.1.2., step 1).
4. Sodium phosphate buffer: 10 mM sodium phosphate buffer, pH 6.5 (adjusted with NaOH).
5. 0.5 M Guanidinium chloride buffer: 0.5 M guanidinium chloride, 5 mM EDTA, 10 mM
sodium phosphate buffer, pH 6.5 (adjusted with NaOH).
2.3. Gel Chromatography
2.3.1. 4 M Guanidinium Chloride Buffer
1. Elution buffer: 4 M guanidinium chloride, 10 mM sodium phosphate buffer, pH 7.0 (can
be stored at room temperature).
2.3.2. Gels and Columns
We use either Sepharose CL-2B or Sephacryl S-500HR (Pharmacia Biotech,
Uppsala, Sweden) for the separation of mucins, reduced mucin subunits, and pro-
teolytic fragments of mucins. Both “whole” mucins and subunits are usually excluded
on Sephacryl S-500, but since Sepharose CL-2B is slightly more porous, mucin sub-
units are included and can often be separated from whole mucins on this gel. In our
experience, whole mucins show a tendency to adhere to Sephacryl gels, which is not
seen with Sepharose gels.
2.4. Ion-Exchange High-Performance Liquid Chromatography
Ion-exchange high performance liquid chromatography is carried out in our labora-
tory using a Mono Q HR 5/5 (Pharmacia Biotech) column and eluants based upon a
piperazine buffer system with lithium perchlorate as the elution salt (see Note 5).
2.4.1. Separation of Reduced Mucin Subunits and Proteolytic Fragments
of Mucins (
see
Note 6).
1. Buffer A: 0.1% (w/v) CHAPS in 6 M urea, 10 mM piperazine/perchlorate buffer, pH 5.0
(adjusted with perchloric acid).
2. Buffer B: 0.1% (w/v) CHAPS in 6 M urea, 0.25–0.4 M LiClO
4
, 10 mM piperazine/per-
chlorate buffer, pH 5.0 (adjusted with perchloric acid).
3. Buffer C: 10 mM piperazine/perchlorate buffer, pH 5.0 (adjusted with perchloric acid).
4. Buffer D: 0.25–0.4 M LiClO
4
in 10 mM piperazine/perchlorate buffer, pH 5.0 (adjusted
with perchloric acid).
6 Davies and Carlstedt
3. Methods
3.1. Extraction of Mucins from Mucous Secretions
1. Thaw secretions, if necessary, preferably in the presence of 1 mM DFP.
2. Mix the secretions with an equal volume of ice-cold PBS containing protease inhibitors.
3. Centrifuge secretions at 4°C in a high-speed centrifuge (23,000g average [av]).
4. Pour off the supernatant, which represents the sol phase.
5. Add 6 M guanidinium chloride extraction buffer to the pellet (which represents the gel
phase) and stir gently overnight at 4°C. If samples are difficult to disperse, the material
can be suspended using two to three strokes in a Dounce homogenizer (Kontes Glass Co.,
Vineland, NJ) with a loose pestle.
6. Centrifuge secretions at 4°C in a high-speed centrifuge (23,000g av).
7. Pour off the supernatant corresponding to the “soluble” gel phase mucins.
8. If necessary, repeat steps 5–7 another two to three times or as long as mucins are present
in the supernatant.
9. Add 6 M guanidinium chloride reduction buffer containing 10 mM dithiothreitol (DTT)
to the extraction residue (equivalent to the “insoluble” gel mucins).
10. Incubate for 5 h at 37°C.
11. Add iodoacetamide to give a 25 mM solution, and incubate overnight in the dark at room
temperature.
12. Centrifuge secretions at 4°C in a high-speed centrifuge (23,000g av).
13. Pour off the supernatant corresponding to the reduced/alkylated “insoluble” mucin
complex.
3.2. Extraction of Mucins from Tissue Samples
Tissue pieces are usually supplied to our laboratory frozen at –20°C. If mucins are
to be prepared from the surface epithelium and the submucosa separately, begin with
step 1. If mucins are to be extracted from the whole tissue, begin with step 4.
1. Thaw the tissue in the presence of 10 mM sodium phosphate buffer, pH 7.0, containing
1mM DFP.
2. Scrape the surface epithelium away from the underlying mucosa with a glass microscope
slide.
3. Place the surface epithelial scrapings in ice-cold 6 M guanidinium chloride extraction
buffer and disperse with a Dounce homogenizer (two to three strokes, loose pestle).
4. Cut the submucosal tissue into small pieces and submerge in liquid nitrogen. Pulverize or
grind the tissue (for this purpose we use a Retsch tissue pulverizer, Retsch, Haan,
Germany).
5. Mix the powdered tissue with ice-cold 6 M guanidinium chloride extraction buffer and
disperse with a Dounce homogenizer (two to three strokes, loose pestle).
6. Gently stir samples overnight at 4°C.
7. Centrifuge secretions at 4°C in a high-speed centrifuge (23,000g av).
8. Pour off the supernatant corresponding to the “soluble” mucins.
9. Repeat steps 5–7 three more times, if necessary.
10. Add 6 M guanidinium chloride reduction buffer containing 10 mM DTT to the extraction
residue.
11. Incubate for 5 h at 37°C.
12. Add iodoacetamide to give a 25 mM solution and incubate overnight in the dark at room
temperature.
Isolation of Large Gel-Forming Mucins 7
13. Centrifuge secretions at 4°C in a high-speed centrifuge (23,000g av).
14. Pour off the supernatant corresponding to the reduced/alkylated “insoluble” mucin
complex.
3.3. Isopycnic Density Gradient
Centrifugation in CsCl/Guanidinium Chloride
3.3.1. Isopycnic Density Gradient Centrifugation
in CsCl/4
M
Guanidinium Chloride
1. Dialyze samples against 10 vol of 6 M guanidinium chloride extraction buffer. The vol-
ume of the sample that can be run in each tube is two-thirds of the total volume held by
the tube.
2. For practical purposes, the preparation of gradients is carried out by weighing rather than
measuring volumes. Check the volume by weighing (the density of 6 M guanidinium
chloride is 1.144 g/mL; see Note 1). If the sample volume is less than two-thirds of the
total, fill up to the required volume with 6 M guanidinium chloride.
3. Weigh the required amount of CsCl to give the correct density into a beaker (see Note 3).
4. Add the sample to the CsCl and stir gently.
5. The final weight of the sample is calculated from the volume of the tube and the final
density of the solution. Add sodium phosphate buffer to give the final weight and stir the
sample gently.
6. Measure the density of the sample prior to loading with a syringe and cannula into the
tubes. Balance the tubes carefully and seal according to the manufacturer’s instructions.
7. Centrifuge the samples. We use a Beckman L-70 Optima centrifuge and either a 50.2Ti
rotor (tube capacity 40 mL), with a starting density 1.39 g/mL, or a 70.1Ti rotor (tube
capacity 13 mL), with a starting density of 1.40 g/mL. Samples are centrifuged at 36,000
rpm (50.2Ti rotor) or 40,000 rpm (70.1Ti rotor) at 15°C for 72–96 h (see Note 7). These
conditions give gradients of approx 1.25–1.60 g/mL but will vary according to the rotor
geometry, starting density, and speed used. Care should be taken to ensure that the start-
ing concentration of CsCl at a given rotor speed and temperature does not exceed that
recommended so that CsCl does not precipitate at the bottom of the tubes during the
centrifugation run. This information should be available in the manufacturer’s rotor
handbook.
8. After centrifugation, recover 20–40 fractions from the gradients by piercing the bottom
of the tubes and collecting fractions with a fraction collector equipped with a drop counter.
Analyze the fractions for density (by weighing a known volume) and absorbance at 280
nm, as well as the appropriate carbohydrate and antibody reactivities.
9. Large amounts of proteins/lipids in the samples may lead to a poor separation between
these molecules and mucins. In this case, mucin-containing fractions may be pooled and
subjected a second time to density gradient centrifugation in CsCl/4 M guanidinium chlo-
ride. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the mucin-containing
fractions may be used to determine whether all proteins have been removed.
3.3.2. Isopycnic Density Gradient Centrifugation
in CsCl/0.5
M
Guanidinium Chloride
Density Gradient Centrifugation in CsCl/4 M guanidinium chloride may be fol-
lowed by subjecting the mucin-containing fractions to a second density gradient step
in CsCl/0.5 M guanidinium chloride, which gives a better separation between mucins
8 Davies and Carlstedt
and DNA (see Note 3). Some mucins show a tendency to precipitate in the presence of
CsCl at low concentrations of guanidinium chloride, and CHAPS is sometimes added
to the gradients to counteract this effect.
1. Dialyze samples against 10 vol of 0.5 M guanidinium chloride buffer.
2. Measure the volume of the sample by weighing (the density of 0.5 M guanidinium chlo-
ride is 1.015 g/mL; see Note 3).
3. Weigh cesium chloride to give the required density into a beaker (see Note 3).
4. Add the sample (volume must not exceed three-fourths of the total volume held by
the tube).
5. If required, add 1% CHAPS solution to give a final concentration of 0.01% (i.e., 1% of
the total volume).
6. The concentration of guanidinium chloride in the final volume must be 0.5 M, and the
volume of the CsCl and CHAPS must therefore be compensated for by the addition of a
small volume of 8 M guanidinium chloride.
7. The final weight of the sample is calculated from the volume of the tube and the final
density of the solution. Add sodium phosphate buffer to give the final weight and stir the
sample gently.
8. Measure the density of the sample and load into the tubes with a syringe and cannula.
Seal the tubes according to the manufacturer’s instructions.
9. Centrifuge the samples at 36,000 rpm (50.2Ti rotor, starting density 1.50 g/mL) or 40,000
rpm (70.1Ti rotor, starting density 1.52 g/mL) at 15°C for 72–96 h (see Note 7). These
conditions give gradients of approx 1.35–1.67 g/mL but will vary according to the rotor
geometry, starting density, and speed used. Care should be taken to ensure that the start-
ing concentration of CsCl at a given rotor speed and temperature does not exceed that
recommended so that CsCl does not precipitate at the bottom of the tubes during the
centrifugation run. This information should be available in the manufacturer’s rotor
handbook.
10. After centrifugation, recover 20–40 fractions from the gradients by piercing a hole in the
bottom of the tubes and collecting fractions with a fraction collector equipped with a drop
counter. Analyze the fractions for density (by weighing a known volume) and absorbance
at 280 nm, as well as the appropriate carbohydrate and antibody reactivities.
3.4. Gel Chromatography
3.4.1. Sepharose CL-2B
1. Elute columns (100 × 1.6 cm) packed according to the manufacturer’s specifications with
4 M guanidinium chloride buffer at a rate well below the maximum of 15 mL/(cm
–2
.
h
–2
).
2. Apply samples, the volume of which should be <5% of the column volume, that have
been dialyzed against the running buffer to the column through an injector.
3. Monitor the eluate on-line with an ultraviolet (UV) monitor and collect fractions using a
fraction collector and subject to the appropriate carbohydrate and antibody analyses.
3.4.2. Sephacryl S-500
1. Elute columns (50 × 1.6 cm) packed according to the manufacturer’s specifications are
eluted with 4 M guanidinium chloride buffer at a flow well below the maximum rate of
40 mL/(cm
–2
.
h
–2
). We run S-500HR columns on a system consisting of a 2150 LKB tita-
nium head pump and a Pharmacia V-7 injector (Pharmacia Biotech).
Isolation of Large Gel-Forming Mucins 9
2. Apply samples, the volume of which should be <5% of the column volume, that have
been dialyzed against the running buffer to the column through an injector.
3. Monitor the eluate on-line with a UV monitor and collect fractions using a fraction col-
lector and subject to the appropriate carbohydrate and antibody analyses.
3.5. Ion-Exchange Chromatography (
see
Note 5)
1. Run Mono Q columns on a system comprising a 2150 LKB titanium head pump con-
nected to a 2152 LKB controller and a Pharmacia V-7 injector. All connections are made
using Teflon tubing.
2. Equilibrate the column with buffer A or C.
3. Dialyze sample exhaustively or dissolve sample in buffer A or C and apply the sample to
the column.
4. Run the column in a linear gradient up to 100% buffer B or D.
5. Monitor the eluate on-line with a UV monitor and collect fractions using a fraction col-
lector and subject to the appropriate carbohydrate and antibody analyses.
3.6. Analysis of Mucins
Methods for the detection and analysis of mucins are dealt with in other chapters in
this volume. However, three principally different methods are available: solution
assays such as colorimetric assays for hexose and sialic acid; membrane-based meth-
ods such as slot-blotting and staining with periodic acid-Schiff reagent; antibodies and
lectins or coating methods such as the glycan detection method and enzyme-linked
immunosorbent assays (ELISA). All these techniques have advantages and disadvan-
tages. Solution methods often crave larger amounts of material than the other two, but
selective loss of components is less of a problem. Membrane-based methods allow
relatively large volumes of “dilute” sample to be analyzed, thus increasing the sensi-
tivity, but components that do not adhere to the membrane may be lost and the linear
range of the technique may be limited. “Coating methods” such as ELISA are prone to
artefacts if samples are concentrated, and care must be taken to ensure that the signals
obtained are within the linear range for the technique.
4. Notes
1. The molarity of guanidinium chloride solution can be calculated from the density accord-
ing to the following formula:
M =( ρ – 1.003)/0.02359
where M is the molarity and ρ is the density in grams per milliliter.
2. Inhibitors are added to the 6 M guanidinium chloride extraction buffer in order to block
the activity of the three major classes of proteolytic enzymes: metalloproteases, serine
proteases, and thiol proteases. The action of metalloproteases is inhibited by the addition
of EDTA to the buffer. This can be added during the initial preparation since it is stable at
room temperature. DFP is a potent inhibitor of serine proteases and esterases, including
acetylcholinesterase, and should therefore be handled in a fume cupboard with extreme
care! DFP is supplied in 1-g vials with a septum, and prior to dilution, vials should be
cooled on ice to reduce the vapor pressure. Under supervision, the septum should be pierced
with a needle to equilibrate the pressure, and the DFP should be transferred using a syringe
and needle. The contents of the vial should be placed directly into the correct volume of
10 Davies and Carlstedt
ice-cold dry propan-1-ol to give a 100 mM solution. DFP is unstable in water but can be
stored at –20°C in propan-1-ol. After dilution, the vial as well as the needles and syringes
used may be rinsed with 1 M NaOH to inactivate the DFP. Phenylmethylsulfonylfluoridate
(PMSF) can be used at a concentration of 0.1 mM in place of DFP. However, we find this
a less attractive option owing to its low solubility although it is possible to prepare first a
stock solution of PMSF in an organic solvent that is miscible with water. Thiol proteases
are inactivated through the addition of NEM. In addition, NEM will also block exchange
reactions between free thiol groups and disulfide bonds.
3. Samples in our laboratory are usually subjected to a two-stage isopycnic density gradient
procedure (see Note 4). First, samples are centrifuged in CsCl/4 M guanidinium chloride,
which gives a good separation of higher buoyant density mucins and nucleic acids from
low buoyant density proteins, glycoproteins, and lipids while maintaining a denaturing
environment. Thus, proteolytic enzymes can be separated from mucins before the con-
centration of guanidinium chloride is reduced. The second step of the purification is to
pool the partially separated mucins and nucleic acids and subject them to a second density
gradient step in CsCl/0.5 M guanidinium chloride. These conditions give a good group
separation between mucins and nucleic acids. The amount of cesium chloride needed to
give a required density in 4 or 0.5 M guanidinium chloride can be calculated according to
the following formula:
x = v (1.347ρ – 0.0318M – 1.347)
where x is CsCl (grams), v is the final volume, M is the molarity of the guanidinium
chloride (4 or 0.5M), and ρ is the density (grams per milliliter).
4. In our laboratory, CsCl rather than CsBr or CsSO
4
, is used as the density gradient–
forming salt since gradients are run in the presence of guanidinium chloride and the use
of CsBr or CsSO
4
in the presence of guanidinium chloride gives rise to mixed cesium
salts. Figure 1 shows a comparison of the separation obtained between mucins and
DNA using the two-step approach in CsCl/4 M guanidinium chloride followed by CsCl/
0.5 M guanidinium chloride with that given by CsBr or CsSO
4
in 10 mM sodium phos-
phate buffer. DNA was mixed with purified cervical mucins and gradients prepared
using each of the cesium salts. In CsCl/4 M guanidinium chloride, there is poor resolu-
tion of mucins from DNA (Fig. 1A); however, a reduction in the concentration of
guanidinium chloride to 0.5 M leads to a baseline separation between mucins and DNA
in this salt (Fig. 1B). In CsSO
4
, mucins are also completely separated from DNA (Fig. 1C).
In CsBr, however, DNA and mucins have a similar buoyant density, and DNA trails
into the mucin peak (Fig. 1D). These data indicate that CsBr is not the salt of choice for
samples containing DNA.
5. Traditionally, we have used lithium perchlorate as the elution salt since it is compatible
with our colorimetric assays for carbohydrate based on sulfuric acid (e.g., the anthrone
procedure). Alternative salt/buffer systems may give at least as good, if not better, sepa-
ration depending on the nature of the mucins in question.
6. The optimum concentration of LiClO
4
in buffers B and D varies between 0.25 and 0.5 M
depending on the charge densities of the mucins to be separated, although typically we use a
concentration of 0.4 M. For buffers A and B, stock solutions of 8 M urea are freshly prepared
and run through a column containing a mixed anion/cation exchanger (e.g., Elgalite or
Amberlite resin). The buffer system A and B containing 6 M urea and 0.1% CHAPS gives
good separation between different populations of reduced mucin subunits, whereas for
the separation of proteolytic fragments, buffers C and D are used.
Isolation of Large Gel-Forming Mucins 11
Fig. 1. Density gradient centrifugation of cervical mucins and DNA. Purified cervical mu-
cins were mixed with DNA and subjected to density gradient centrifugation in (A) CsCl/4 M
guanidinium chloride; (B) CsCl/0.5 M guanidinium chloride; (C) CsSO
4
/10 mM sodium phos-
phate buffer, pH 6.5; and (D) CsBr/10 mM sodium phosphate buffer, pH 6.5. After centrifuga-
tion in a Beckman L70 centrifuge (70.1Ti rotor, 40,000 rpm, 15°C, 65 h, starting density:
[A] 1.41 g/mL, [B] 1.52 g/mL, [C] 1.34 g/mL, and [D] 1.49 g/mL), fractions were collected
from the bottom of the tubes and analyzed for sialic acid (
᭹
), carbohydrate (glycan detection
method) (
ᮀ
), MUC5B antibody reactivity (
᭜
), absorbance at 280 nm (
), and density (
).
12 Davies and Carlstedt
7. The rotor type, speed and starting densities rather than the g-force are given for the runs.
In our experience, the rotor geometry (the tube angle within the rotor), starting density,
and the speed at which the run is conducted are the most important factors determining
the gradient formed and the conditions cannot necessarily be reproduced by using the
same g-force in another rotor.
Acknowledgments
The authors acknowledge support from the Swedish Medical Research Council
(grant no. 7902, 9823), the Medical Faculty of Lund, Centrala Försökdjursnämnden,
Swedish Match AB, the Swedish Cancer Fund, Greta och Johan Kocks Stiftelse,
Stiftelsen Riksförbundet Cystisk Fibros, Vårdals Stiftelse, and the Smokeless Tobacco
Research Council, Inc. (USA).
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