Cell Surface Biotinylation and Other
Techniques for Determination of
Surface Polarity of Epithelial
A fundamental property of epithelial cells is the
polarized distribution of proteins and lipids in the
apical and basolateral domains of the plasma membrane.
These two domains are physically separated
from each other by the tight junction. Many studies
have been done over the past 20 years to understand
the mechanisms that lead to the establishment and
maintenance of the polarized distribution of proteins
and lipids in the plasma membrane of epithelial cells
(Rodriguez-Boulan et al.
, 2005; Yeaman et al.
Keller and Simons, 1997).
A major advance in the study of epithelial cell polarity
was achieved with the introduction of porous filter
supports for the growth of epithelial cell cultures
(reviewed in Rodriguez-Boulan et al.
, 2005). This
method differs from classical cell culture in that it
allows direct access to the basolateral surface of cultured
cells. Epithelial cells and cell lines grown on such
filter supports (either nitrocellulose or polycarbonate)
attain a more differentiated appearance and become
polarized after relatively short times in culture. While
most studies on epithelial polarity and trafficking of
plasma membrane proteins have been performed
using a limited number of cell lines (MDCK, FRT,
Caco-2), this culture technique has been gaining in
popularity and has been used now for primary cultures as well. The confluency of cells grown on permeable
supports can be determined by the measurement
of transepithelial electrical resistance or
H]inulin flux (Hanzel et al.
One of the biggest advantages of this culture system
is the accessibility of either the apical or the basolateral
surface to any reagent added to the medium
and the ability to add different reagents to contact
either surface. This is the basis of the biotinylation
techniques that have been developed to selectively
label proteins present on the apical or basolateral
domains of the plasma membrane of filter-grown
The proteins present on the surface of filter-grown
monolayers can be selectively modified by the watersoluble
cell-impermeable biotin analog sulfo-NHSbiotin.
Taking advantage of the access afforded by the
filter support, the addition of sulfo-NHS-biotin to only
one surface of the cell results in the selective labeling
of only the apical or basolateral surface proteins. The
biotinylated proteins can then be detected by blotting
I]streptavidin or streptavidin conjugated to
any number of enzymatic reporters. Furthermore,
the cells can be metabolically pulse labeled and the
proteins of interest can then be studied using biotinylation,
immunoprecipitation and subsequent streptavidin-
agarose precipitation. This technique is very
versatile and is applicable to the study of diverse
aspects of epithelial cell polarity, such as the steadystate distribution of specific antigens, or dynamic
processes, such as targeting to the cell surface and
transcytosis of membrane proteins. Several biotin
analogs are available, including one that contains a
disulfide bond. By differentially labeling the surfaces
of epithelia in situ
with the cleavable NHS-S-S-biotin
and a noncleavable biotin, we have been able to study
the polarity of a native epithelium in situ
(Marmorstein et al.
The first edition of this article described a basic protocol
for selective cell surface biotinylation, plus some
modifications of the assay to study protein targeting
and endocytosis. The second edition included a basic
protocol for in situ
domain-specific biotinylation. In
this edition, we have added a protocol of surface
immunolabeling as an alternative to biotin labeling for
determination of polarity. This technique is critical in
cell lines that may exhibit leakiness to biotin. Additionally,
we have also included two sections determining
the polarity of a protein by means of intranuclear
microinjection of its cDNA and quantitative microscopic
analysis to determine distribution of the protein
relative to known polarized markers.
Sulfo-NHS-biotin (sulfosuccinimidobiotin, Cat. No.
21217); NHS-LC-biotin (sulfosuccinimidyl-6-(biotinamido)-
hexanoate, Cat. No. 21335); NHS-SS-biotin
Cat. No. 21331); and immunopure-immobilized
streptavidin (Cat. No. 20347) are from Pierce
(Rockville, IL). Protein A-Sepharose Cl-4B (Cat. No.
17-0780-01) is from Pharmacia/LKB (Piscataway, NJ).
Glutathione (Cat. No. G-6529) and cycloheximide (Cat.
No. C-7698) are from Sigma Chemical Co. (St. Louis,
MO). Staphylococcus aureus
cells (Pansorbin, Cat. No.
507858) are from Calbiochem (La Jolla, CA). Cells are
grown on polycarbonate filters (Transwell, 12mm
diameter, Cat. No. 3401; 24mm diameter, Cat. No.
3412) from Corning-Costar (Cambridge, MA); MEMSelect-
Amine kits (Cat. No. 19050-012) are from GIBCO
BRL Life Technologies (Grand Island, NY);
S (methionine/cysteine) and [35
are from Dupont NEN (Boston, MA) (Cat. No.
NEG 072 for Express and Cat. No. NEG 022T for
I]streptavidin can be obtained from
Amersham (Arlington Heights, IL) (Cat. No. IM236);
streptavidin conjugated to horseradish peroxidase can
be obtained from Sigma (Cat. No. S-5512).
A. Cell Surface Biotinylation
This procedure is used to determine the relative percentage
of a plasma membrane protein(s) in the apical
versus basolateral plasma membrane of epithelial cells
grown on permeable filter supports (modified from
Sargiacomo et al.
- PBS-CM: Phosphate-buffered saline containing
1.0 mM MgCl2, and 1.3 mM CaCl2
- Sulfo-NHS-biotin or sulfo-NHS-LC-biotin: Stock
solution is 200mg/ml in dimethyl sulfoxide (DMSO),
which can be stored for up to 2 months at -20°C. Thaw
just prior to use and dilute to a final concentration of
0.5 mg/ml in PBS-CM. Use immediately.
- 50mM NH4Cl in PBS-CM or Dulbecco's modified
Eagle's medium (DMEM): Use to quench the excess
biotin at the end of the labeling reaction
All steps are carried out on ice and with ice-cold
Analysis of Results
- For all experiments, use confluent monolayers of
cells plated at confluency (for most cell lines, 2.5-3.5 × 105 cells/cm2 of filter) 4-5 days prior to biotinylation.
Measure transepithelial electrical resistance (TER) and
discard monolayers that do not exhibit acceptable
resistances (different cell lines exhibit different TER
values ranging from tens to thousands of Ω · cm2;
monolayers should be used that exhibit TER values in
the normal range for your cell line. We have successfully
performed this assay on cells with TERs as low
as 50 Ω · cm2).
- Wash filters on both sides three times with icecold
- Add a fresh solution of sulfo-NHS-biotin
(0.5mg/ml in PBS-CM) to the apical or basolateral
chamber. Add PBS-CM to the other chamber. We
use 0.7ml apical and 1.4ml basolateral for 24-mmdiameter
filters and 0.4 and 0.8 ml for 12-mm-diameter
filters. Incubate with gentle shaking for 20min at 4°C and then repeat this step.
- Quench the reaction by removing the solutions
from both chambers and replacing with 1 ml of 50 mM NH4Cl in PBS-CM. Incubate with gentle shaking for
10min at 4°C.
- Rinse twice with PBS-CM.
- Excise filters and either freeze at -80°C (the
freeze thaw involved with storage at -80°C appears to inactivate some proteases) or immediately proceed
with the extraction of biotinylated proteins as outlined
in Section III,F.
The amount of protein present on the apical or basolateral
surface is determined by a densitometric analysis
of the autoradiographs. Multiple exposures are
necessary if using the film to ensure that the values
obtained are in the linear range of the film. Polarity is
expressed as the percentage of total surface protein
present on one surface of the monolayer.
B. Biotin Targeting Assay
This procedure is used to determine if proteins are
delivered directly, indirectly (transcytotically), or nonpolarly
to the apical and/or basolateral surface of an
epithelial cell (modified from Le Bivic et al.
- Starvation medium: DMEM without methionine
or cysteine. This solution is prepared using a MEM
Select-Amine kit by not adding the methionine and
- [35S]EXPRESS (methionine/cysteine:) or [35S]cysteine:
1 mCi per multiwell plate (12 x 1.2-cm or 6 x
2.4-cm-diameter filters). In some cases, proteins are
effectively labeled with [35S]SO4, an advantage for
studies of post-Golgi sorting, because the addition of
sulfate occurs in the trans-Golgi network (see chapter
by Kreitzer et al.).
- Chase medium: DMEM containing a 10x concentration
of methionine and cysteine (made by addition
of methionine and cysteine to starving medium) or the
normal medium in which the cells grow.
- HCO3-free DMEM containing 20mM HEPES and
0.2% bovine serum albumin (BSA)
- Sulfo-NHS-biotin (NHS-LC-biotin or NHS-SSbiotin):
0.5 mg/ml in PBS-CM + all of the reagents used
in the cell surface biotinylation protocol.
- Lysis buffer: 1% Triton X-100 in 20mM Tris,
150mM NaCl, 5mM EDTA, 0.2% BSA, pH 8.0, and
- Immunopure-immobilized streptavidin on agarose
- 10% SDS: sodium dodecyl sulfate
C. Targeting Assay by Surface Immunolabeling
- Wash cells on filters three times with starvation
medium and incubate for 20-40min in starvation
medium. The starving period will depend on your cell
type. MDCK cells work well with a 20-min starvation;
RPE-J require longer times.
- Pulse for 20-30min (again depends on cell line)
in starving medium containing [35S]EXPRESS of
[35S]cysteine at 37°C. The pulse solution is starving
medium plus the 35S label. Minimal volumes are recommended.
For MDCK cells we pulse with 20-40µl
from the basolateral surface. A drop of pulse medium
is placed on a strip of Parafilm in a humidified
chamber (we use a plastic box lined with wet towels),
and the insert containing the filter and MDCK monolayer
is removed from the multiwell and dropped on
top of the pulse medium. Some cell lines (i.e., RPE-J)
are better labeled from the apical surface. For apical
pulse, starvation medium is removed from both chambers
and pulse medium is applied only to the apical
chamber. For 1.2-cm-diameter filters we use a 100-µl
volume; for 2.4-cm-diameter filters we use a 350-µl
volume of pulse medium.
- The pulse is terminated by washing with chase
medium three times.
- At different chase times, aspirate chase medium
and replace with ice-cold NaHCO3-free DMEM containing
20mM HEPES and 0.2% BSA and store on ice
until all chase points have been collected.
- Proceed to apical or basolateral biotinylation following
the protocol described earlier for cell surface
- Excise filters and either freeze at -80°C or immediately
lyse cells and immunoprecipitate specific
proteins as described in the section extraction of
- Remove immunoprecipitated proteins from
beads by adding 40µl of 10% SDS and heating for
5 min at 95°C. Immediately dilute with 460µl of lysis
buffer and pellet for 1 min in a microfuge. Remove
450µl and place in a new tube. Dilute the remaining
50µl with 50µl of 2x Laemmli sample buffer. This
sample is used to normalize for differential incorporation
of radiolabel from filter to filter. The remaining
450µl is diluted with a further 1 ml of lysis buffer to
which is added an additional 50µl of streptavidin
agarose that has been preblocked for 1-12h with lysis
- Streptavidin precipitation is allowed to proceed
for 1 h to overnight at 4°C. Then the beads are washed
successively in TPII, TPIII, and TPIV as described in
Section III,E After the final wash the beads are resuspended
in Laemmli sample buffer and heated to 95°C for 5 min.
- Both the sample representing total and surface
protein are resolved on SDS-PAGE gels. The gels are
dried and exposed for autoradiography.
Epithelial cells such as LLC-PK1 may not form a
tight monolayer and hence could be leaky to biotin
analogs (MW~ 400-600). To overcome this problem,
we have used antibody labeling against the protein of
interest (Gan et al.
, 2002). Antibodies are less likely to
traverse through leaky monolayers. Leakiness of antibodies
should be directly determined before using this
- PBS-CM: See Section III,A
- DMEM containing 0.2% BSA
Analysis of Results
- The initial steps are similar to Section III,B (steps
1-4). Label the ice-cold filters from different chase time
points with antibody added to either apical or basolateral
domains for 1 h on ice in a cold room kept at
4°C. Dilute the antibody in DMEM containing 0.2%
BSA at an approximate concentration of 1 µg/ml. After
1 h, wash filters four times in ice-cold PBS-CM containing
- Excise filters and either freeze at -80°C or immediately
lyse cells in lysis buffer.
- Pull down the antigen-antibody complex with
protein A or G beads from nine-tenths of the postnuclear
supernatants. Subject one-tenth of the supernatant
again to immunoprecipitation to measure total
- Wash immunoprecipitates on the beads successively
in TPII, TPIII, and TPIV as described in Section
- After the final wash, resuspend the beads in
Laemmli sample buffer and heat to 95°C for 5 min.
- Resolve both the sample representing total and
surface proteins on SDS-PAGE gels. Dry and expose
the gels for autoradiography.
The polarity of the protein is determined at each
time point by densitometric analysis of the autoradiographic
data. The values obtained for the surface
protein should be normalized against the values
obtained form the totals (including precursor forms).
This controls for differences in the incorporation of
label (specific activity) between monolayers. If the
protein is highly polarized from the first time point at
which it is detected on the cell surface, then it is delivered
directly to that surface. If it is polarized on one
surface early in the chase and then switches polarity
later in the chase, then it is delivered indirectly. If the protein is nonpolar early in the chase and acquires
polarity only after longer chase times, then it is not
sorted in the TGN, but its final polarity is acquired by
differential stability on the apical and basolateral
D. Biotin Assay for Endocytosis
This assay examines the internalization of plasma
membrane proteins (from Graeve et al.
- Cleavable biotin reagent: NHS-SS-biotin
- DMEM containing 0.2% BSA
- Reducing solution: 310mg glutathione (free acid)
dissolved in 17ml H2O (50mM). Add 1 ml of 1.5M NaCl, 0.12ml of 50% NaOH, and 2ml of serum just
- Quenching solution: 5mg/ml iodoacetamide in
PBS-CM containing 1% BSA
Analysis of Results
- Wash cells on filters four times, 15 min each time
with ice-cold PBS-CM.
- Add 1 ml of NHS-SS-biotin (0.5mg/ml in icecold
PBS-CM) to the chamber being labeled and PBSCM
to the other chamber. Incubate for 20min at 4°C and repeat with fresh solutions.
- Wash filters twice with DMEM/0.2% BSA. Keep
two filters on ice (one of these will represent the total
amount of proteins at the surface before internalization
and the other will be treated with the reducing
solution and represents your control of efficiency of
reduction) and transfer the other filters to 37°C for
various times to allow the biotinylated proteins to be
- Stop incubation by transferring filters back to
- Wash twice in PBS-CM + 10% serum.
- Incubate filters for 20min in reducing solution.
Repeat. (Mock treat one filter.)
- After washing, quench free SH groups in
5mg/ml iodoacetamide in PBS-CM + 1% BSA for
- Lyse cells and immunoprecipitate as described in
the section extraction of biotinylated proteins.
- Run the samples on SDS-PAGE gels, transfer to
nitrocellulose or PVDF, blot with [125I]streptavidin, and
expose for autoradiography.
Endocytosis of the protein of interest is indicated
by protection of the NHS-SS-biotin-labeled surface protein from reduction by glutathione. By chasing the
cells for various lengths of time, a rate of endocytosis
can be calculated by comparing the percentage of
protein protected at each time point. Obviously if none
of the protein is protected from reduction, it is 100% at
the surface; conversely, if all of the protein is protected,
100% has been internalized.
E. In Situ Domain Selective Biotinylation of
Retinal Pigment Epithelial Cells
The retinal pigment epithelium is uniquely suited
for biochemical studies of polarity in situ
. The RPE
exists as a natural monolayer with a broad apical
surface that is easily exposed after gentle enzymatic
treatment to remove the adjacent neural retina. The
apical surface of the tissue is labeled with a noncleavable
biotin analog such as NHS-LC-biotin for the
identification of apical proteins. For identification
of basolateral proteins the biotinylatable sites on the
apical surface are labeled with the cleavable NHS-SSbiotin.
After isolation of RPE cells, the nonlabeled
basolateral proteins are labeled in suspension with the
noncleavable form. Removal of the cleavable NHS-SSbiotin
by reduction with 2-mercaptoethanol results in
the presence of biotin only in the population of proteins
present on the basolateral surface of the RPE
(Marmorstein et al.
- Sulfo-NHS-biotin, or NHS-LC-biotin, and NHS-SSbiotin
stock solutions in DMSO
- HBSS: 10mM HEPES buffered Hank's balanced
- DMEM containing 10 mM HEPES
- Bovine testicular hyaluronidase
- CMF-PBS: PBS calcium and magnesium free
- PBS-EDTA: CMF-PBS + 1 mM EDTA
All steps are carried out on ice unless otherwise
Analysis of Results
- Rats are euthanized by CO2 asphyxiation, and
the eyes are enucleated and stored for 3 h to overnight
in the dark on ice in HBSS.
- A circumferential incision is made above the ora
serrata, and the cornea, iris, lens, and vitreous are
- The eyecups are incubated for 10-30 min at 37°C in HBSS containing 290 units/ml bovine testicular
- The ora serrata is removed, and the neural retina
is peeled carefully away from the RPE. The optic nerve
head is severed and the neural retina is removed. The
RPE is inspected under the dissecting microscope.
Black spots on the outer surface of the retina or tracts
of smooth reflective surface in the eyecup indicate
damage. Damaged eyecups are discarded.
- Soluble components of the interphotoreceptor
matrix are removed by incubation in 2-ml microcentrifuge
tubes (one eye per tube) on a rotator in ice-cold
HBSS for 20min. This is repeated three times.
- The apical surface of the RPE in one eyecup is
biotinylated with 1 ml of PBS-CM containing 2mg of
sulfo-NHS-biotin. The other eyecup is biotinylated
with 1 ml of PBS-CM containing 2mg of NHS-SSbiotin.
This procedure is repeated three times.
- The reaction is quenched with 1 ml of 10mM HEPES buffered DMEM for 10min at 4°C.
- The eyecups are rinsed once in ice-cold CMF-PBS
and are then incubated in PBS-EDTA on ice for 30 min.
- The RPE is gently teased from the inner surface
of the eyecup using a 22-gauge needle. The RPE layer
is collected in a 1.5-ml microcentrifuge tube and pelleted
for 10 s in a microfuge. Cells labeled apically with
noncleavable sulfo-NHS-biotin or NHS-LC-biotin at
this stage are held on ice until the basolateral samples
- For cells labeled with cleavable NHS-SS-biotin,
the pellet is resuspended in 1 ml of PBS-CM containing
2mg/ml sulfo-NHS-biotin or NHS-LC biotin and
incubated on a rotator at 4°C. After 20min the cells
are pelleted for 10s in a microfuge and this step is
- The reaction is quenched with 50 mM NH4Cl in
PBS-CM for 10min. The cells are then pelleted in the
microfuge for 10 s.
- At this point, both apical and basolaterally
labeled pellets are frozen dry at -80°C or immediately
lysed and specific proteins immunoprecipitated as
described in the section extraction of biotinylated
- Immunoprecipitated proteins are resuspended
in Laemmli sample buffer containing 5% 2-
mercaptoethanol or 50mM dithiothreitol to release
the NHS-SS-biotin from the apical proteins in basolateral
samples. After heating to 95°C for 5 min, samples
are resolved by SDS-PAGE, transferred to nitrocellulose
or PVDF membranes, and blotted with
Analysis of the results proceeds as in the section on
cell surface biotinylation.
F. Extraction and Immunoprecipitation of
- TPI: 1% Triton X-100, 20mM Tris, 150mM NaCl,
5 mM EDTA, pH 8.0, containing 0.2% BSA, and protease
- TPII: 0.1% SDS, 20mM Tris, 150mM NaCl, 5 mM EDTA, pH 8.0, containing 0.2% BSA
- TPIII: 20mM Tris, 500mM NaCl, 5 mM EDTA,
pH 8.0, containing 0.2% BSA
- TPIV: 50mM Tris, pH 8.0
- Laemmli sample buffer
- Protein A-Sepharose
- Staphlycoccus A cells: Pansorbin
G. Streptavidin Blotting
- Excise filters from inserts using a #11 scalpel
blade or razor blade. Lyse in 1 ml of TPI at 4°C. In some
cases it may be necessary to use more stringent conditions
for lysis (i.e., RIPA buffer, which contains 0.5%
deoxycholate and 0.1% SDS in addition to 1% Triton X-
- Wash 100 µl/sample of Pansorbin three times
with TPI. Do not omit protease inhibitors.
- Centrifuge lysate in a microfuge at 4°C at
13,000g for 10min. Collect the supernatant and
discard the pellet.
- Add 100µl of washed Pansorbin to each supernatant.
Preclear for 1h at 4°C.
- Resuspend 5-10 mg of protein A-Sepharose/
lysate in 1 ml TPI /lysate. If you are immunoprecipitating
with a mouse IgG, after 10min, when the
Sepharose beads are swollen, add 2mg of rabbit antimouse
IgG. After 1 h wash the beads three times with
TPI. On the last wash, pellet the beads in microcentrifuge
- Pellet the Pansorbin by centrifugation at 13,000g for 10min. Collect the supernatant, add an appropriate
volume of antibody, and incubate at 4°C for 1 h to
- Transfer the immunoprecipitates to the tubes
containing the protein A-Sepharose and incubate for
1 h at 4°C.
- Centrifuge the immunoprecipitates in a
microfuge at 13,000g for 30s. Remove the supernatant
and resuspend the beads in 1 ml of TPI. Repeat this
step three times with TPII, three times with TPIII, and
once with TPIV.
- Resuspend with an appropriate volume of
Laemmli sample buffer, heat to 95°C for 5min, and
resolve by SDS-PAGE. For streptavidin blotting, transfer
the gel to nitrocellulose or PVDF.
- Blocking buffer: 5% Carnation instant milk, 0.3%
BSA, in PBS-CM
- Rinse buffer: 1% BSA, 0.2% Triton X-100 in PBSCM
- [125I]Streptavidin or streptavidin conjugated to horseradish
peroxidase or alkaline phosphatase (streptavidin-
- A phosphorimager, Kodak X-OMAT AR film and
a cassette with an intensifying screen, or an enhanced
chemiluminescence reagent kit (such as those supplied
by Amersham) and appropriate film.
H. Quantitation of Polarized Distribution by
Confocal Microscopy Imaging Technique
- After transfer to nitrocellulose or PVDF (PVDF is
superior for chemiluminescent detection systems and
is used in our laboratory for most streptavidin blots),
block the blot for 1 h in blocking buffer.
- Rinse once with rinse buffer.
- Incubate for 1 h in 40ml rinse buffer containing
1-2 × 106 cpm/ml of [125I] streptavidin or 0.5-
1.0 mg/ml streptavidin-HRP.
- Wash three to five times for 5-10min each with
- Dry the blot and expose to a phosphorimager
screen, autoradiograph it with Kodak X-OMAT AR
film, or use any of the many enhanced chemiluminescent
kits that are available.
Modern quantitative optical microscopy offers an
alternative method to quantify the relative polarity of
fluorescently labeled cell surface proteins in cells
developing polarity in settings such as the calcium
switch assay (Rajasekaran et al.
, 1996). This quantitation
is based on the relative fluorescent pixel intensities
in serial horizontal confocal sections of the target
protein to a known polarized marker.
- Confocal laser-scanning microscope
- Software to measure fluorescence intensity, e.g.,
Metamorph, Image Space Software.
Analysis of Results
- Samples subjected to immunofluorescence technique
(not discussed here) are imaged on a laser confocal
microscopic system. Samples are subjected to optical sectioning (xy) of the entire thickness (z) of the
monolayer. We select an interval between sections for
optimized collection of fluorescence from a given
plane without contribution from the neighboring z planes. Choice of interval depends on pinhole dimension,
which in turn depends on characteristics of the
excitation wavelength. [Refer to a handbook on confocal
microscopy, e.g., Pawley (1995), or a confocal
microscope manufacturer's manual for optimizing
- For each optical section, quantify the average
per-pixel fluorescence intensity of the labeled proteins
using the imaging software. Determine the ratio of
intensity obtained for the protein of interest to that of
a known marker.
The ratio of fluorescent intensity obtained represents
the relative distribution of the target protein and
is interpreted as follows.
I. Determination of Plasma Membrane Protein
Polarity after Intranuclear Microinjection of
- A constant pixel intensity ratio for all optical
sections of a given monolayer suggests overlapping
distribution of the target protein with the known
- Decreasing pixel intensity ratio in from basolateral
to apical domain of the target protein and the
apical or tight junction marker would mean that the
protein is localized in the basolateral domain.
- Increasing intensity ratio with a basolateral
marker would mean that the target protein is apically
targeted (see Rajasekaran et al., 1996).
This procedure is used for rapid qualitative determination
of the steady-state polarity of a newly synthesized
protein in polarized cells. Analysis is
performed using either a fluorescence wide-field or a
confocal microscope. We have utilized this technique
to study the regulation of polarity of basolateral
and apical membrane markers by GTPases and their
downstream effectors. Normal polarization of the
monolayer needs to be confirmed by studying the
localization of known apical or basolateral markers.
- Microinjection buffer: H-KCl buffer containing
10 mM HEPES, pH 7.4, 140 mM KCl. Dissolve 1.04 g
of KCl in 99ml deionized H2O and then add 1.0ml
HEPES from a 1M stock (pH 7.4). Sterilize the buffer
by passing through a 0.22-µm filter and store at 4°C (Müsch et al., 2001).
- PBS/CM: See Section III,A
- H-DMEM: DMEM containing HEPES. Dissolve
bicarbonate-free powdered DMEM in 900ml deionized
H2O. Add 20ml HEPES from a 1M stock and
adjust the pH to 7.4. Sterilize the medium through a
0.22-µm filter and store at 4°C.
- B-DMEM: DMEM containing sodium
Analysis of Results
- Plate MDCK II cells on sterile glass coverslips at
a concentration of 1.6 × 106 cells/ml or at an approximate
plating density of 2 × 105 cells per cm2 in BDMEM
and 10% fetal bovine serum (FBS). Allow the
cells to polarize for 4-5 days and change the medium
only once on day 2 postplating. Growth conditions
required to attain polarity for different cell lines vary
and require optimization.
- Dilute the stock of cDNA (stock prepared in
deionized H2O at a concentration of 0.5mg/ml) in
microinjection buffer to a concentration of around
10µg/ml. It is highly recommended that cDNA constructs
also contain a tag sequence, such as Myc, HA,
GFP, and its variants, in-frame with the gene of interest
so as to distinguish the newly synthesized proteins
from endogenous proteins. For experiments involving
more than one cDNA construct, cDNAs can be coinjected.
However, the efficiency of expression of a construct
may vary in the presence of another. Therefore,
proper conditions should be established for good
expression of each coinjected construct. A range of concentrations
between 1 and 20 µg/ml of DNA should be
tested to optimize their expression levels.
- Prepare microinjection needles by pulling 1-mmdiameter
and 6-in.-long borosilicate glass capillaries
(1B100F-6, World Precision Instruments, Inc, Sarasota,
FL) using a micropipette puller (e.g., Flaming/Brown
Micropipette Puller Model P-97, Sutter Instrument Co.,
- Load the cDNA diluted in microinjection buffer
through the blunt end of the needle into the needle
holder of the micromanipulator (Narishige Company,
Ltd., SE-TAGAYA-KU, Tokyo, Japan) attached to the
inverted microscope (Zeiss-Axiovert 25, Germany).
- Transfer coverslip into 35-mm-diameter tissue
culture dishes. Add 4ml of H-DMEM containing 5%
FBS to each dish and place the dish on the dish holder
of the micromanipulator-microscope described earlier.
Microinject the nucleus of cells. Avoid microinjecting
cells that are right next to each other. This simplifies the analysis of distribution of apical and basolateral
markers. In order to avoid unsynchronized protein
synthesis, preferably microinject within 10-15 min of
transferring the dish to the microscope stage.
- Incubate the microinjected cells with BDMEM-
10% FBS medium at 37°C. Most of the proteins
accumulate in the ER within 60 x 90min at 37°C postmicroinjection.
Different levels of expression should be
tested to ensure that the sorting pathways are not saturated.
For coinjections, it is necessary to standardize
the conditions for sufficient expression of each protein.
Adjustment of DNA concentrations (as described in
step 2) and time of incubation at 37°C postmicroinjection
are two steps that need to be tuned for expression
of multiple constructs.
- After appropriate incubation at 37°C, replace
medium with B-DMEM-10% FBS containing 100 µg/ml
cycloheximide (concentration may be lowered down
to 20µg/ml if cells detach from coverslip) to inhibit
new protein synthesis, the chase time for plasma
membrane delivery of protein is initiated at 37°C for
- After an appropriate chase period, fix cells with
either -20°C chilled methanol for 10min or 2%
paraformaldehyde at room temperature for 15min.
Methanol fixation should be followed by a blocking
step at room temperature with 1% BSA prepared
in PBS-CM for 30min. Paraformaldehyde fixation of
cells is followed by permeabilization at room temperature
for 30min with either 0.2% Triton-X 100 or
0.075% saponin prepared in PBS-CM containing
1% BSA. Cells can now be processed for immunofluorescence
with the appropriate primary and secondary
Cells processed for immunofluorescence are imaged
on either a confocal or a wide-field microscope. The
correct orientation of the cells is determined by analyzing
the staining of known polarized markers. In
case of a wide-field microscope, the entire monolayer
is subjected to z
sectioning at least at 0.5-µm intervals
with a 60x 1.4 NA objective, and standard deconvolution
software is used to enhance the resolution. Alternatively,
we use a confocal microscope in frame-scan
mode and collect xyz
stacks of the entire monolayer
and display the xyz
stack in the orthogonal plane. The
cells can be displayed directly as a xz
cross section by
doing a line scan, i.e., scanning in the xzy
of the protein is determined depending on
the staining pattern, e.g., relative to a tight junctional
marker such as ZO-1.
The methods described here represent examples
of applications of the biotinylation technique; other
examples of possible applications are (1) a transcytotic
assay using a combination of the targeting and endocytosis
protocols (Le Bivic et al.
, 1989; Zurzolo et al.
1992) and (2) detection of GPI-anchored proteins at the
cell surface using Triton X-114 phase separation and
PI-PLC digestion in place of the standard lysis procedure
(Lisanti and Rodriguez-Boulan, 1990). Another
analog of biotin, biotin hydrazide, can be used to label
oligosaccharides of surface glycoproteins following
periodate oxidation (Lisanti et al.
- It has been suggested that the use of pH 9.0
buffer to dilute sulfo-NHS-biotin would enhance the
efficiency of labeling of surface proteins (Gottardi and
Caplan, 1993). In our experience this is not always true
and depends on different proteins and cell lines.
- Always cut the filters out of the plastic holder
before lysis. We have found that the cells can grow
along the inside of the plastic ring supporting the filter.
Lysis of these cells can result in erroneous results
(Zurzolo and Rodriguez-Boulan, 1993).
- Occasionally, in targeting experiments, intracellular
nonbiotinylated forms are recovered on streptavidin
beads. Using NHS-LC-biotin and keeping the
SDS concentration at 0.4% helps reduce this. Another
approach is to use NHS-SS-biotin and remove it from
the streptavidin beads by incubation in 50mM dithiothreitol
of 5-10% 2-mercaptoethanol in 62.5 mM Tris,
pH 8.0. Then spin the beads out and dilute the supernatant
1:1 with 2x Laemmli sample buffer.
- The in situ biotinylation assay works best for proteins
that are restricted to the RPE cell (i.e., RET-PE2
antigen). Quantification of proteins present in adjacent
tissues (particularly the choroid) can contaminate the
basolaterally labeled material and yield an incorrectly
high level of basolateral labeling.
- Determination of polarity by confocal
microscopy can provide visual validation of the biochemical
assay. It is useful for measuring the polarity
of steady-state protein and dynamic changes involved
in tight junction assembly in the Ca2+ switch assay. A
thorough analysis with known markers of different
domains is recommended before determining polarity
of the target protein.
- For determination of polarity by means of
intranuclear microinjection of cDNA, thorough optimization
regarding the protein expression level and
the incubation time is necessary. Moreover, it is critical
to avoid saturating the sorting pathway. Hence, coinjecting
and monitoring a known apical or basolateral
marker are critical for final evaluation. Because the
basolateral domain of the polarized monolayer on the
coverslip is not accessible to the antibody without permeabilization,
it is difficult to distinguish the pool of
basolateral protein that is associated with submembrane
structures closely juxtaposed to the cytoplasmic
side of plasma membrane and the pool present in the
external leaflet of the plasma membrane.
Gan, Y., McGraw, T.E., and Rodriguez-Boulan, E. (2002). The epithelial-
specific adaptor APIB mediates post-endocytic recycling to
the basolateral membrane. Nature Cell Biol
Gottardi, C., and Caplan, M. (1993). Cell surface biotinylation in the
determination of epithelial membrane polarity. J. Tissue Culture
Graeve, L., Drickamer, K., and Rodriguez-Boulan, E. (1989). Functional
expression of the chicken liver asialoglycoprotein receptor
in the basolateral surface of MDCK cells. J. Cell Biol
Hanzel, D., Nabi, I. R., Zurolo, C., Powell, S. K., and Rodriguez-
Boulan, E. (1991) New techniques lead to advances in epithelial
cell polarity. Semin. Cell Biol
Keller R Simons K. (1997). Post-golgi biosynthetic trafficking. J. Cell
Laemmli, U. K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227
Le Bivic, A., Real, E X., and Rodriguez-Boulan, E. (1989). Vectorial
targeting of apical and basolateral plasma membrane proteins in
a human adenocarcinoma cell line. Proc. Natl. Acad. Sci. USA 86
Le Bivic, A., Sambuy, Y., Mostov, K., and Rodriguez-Boulan, E.
(1990). Vectorial targeting of an endogenous apical membrane
sialoglycoprotein and uvomorulin in MDCK cells. J. Cell Biol
Le Gall, A., Yeaman, C., Muesch, A., and Rodriguez-Boulan, E.
(1995). Epithelial cell polarity: New perspectives. Semin. Nephrol
Lisanti, M., Le Bivic, A., Sargiacomo, M., and Rodriguez-Boulan, E.
(1989). Steady state distribution and biogenesis of endogenous
MDCK glycoproteins: Evidence for intracellular sorting and
polarized surface delivery. J. Cell Biol
Lisanti, M., and Rodriguez-Boulan, E. (1990). Glycosphingolipid
membrane anchoring provides clues to the mecanism of protein
sorting in polarized epiothelial cells. Trends Biochem. Sci
Marmorstein, A. D., Bonilha, V. L., Chiflet, S., Neill, J. M., and
Rodriguez-Boulan E. (1996). The polarity of the plasma membrane
protein RET-PE2 in retinal pigment epithelium is developmentally
regulated. J. Cell. Sci
Mfisch A., Cohen D., Kreitzer G., and Rodriguez-Boulan E. (2001).
cdc42 regulates the exit of apical and basolateral proteins from
the trans-Golgi network. EMBO J
Pawley J. B. (ed.) (1995). "Handbook of Biological Confocal
Microscopy." Plenum Press, New York.
Rajasekaran, A.K., Hojo, M., Huima, T., and Rodriguez-Boulan, E.
(1996). Catenins and zonula occudens-1 form a complex during
early stages in the assembly of tight junctions. J. Cell Biol
Rodriguez-Boulan, E., Kreitzer, G., and Muesch, A. (2005). Organization
of vesicular trafficking in epithelia. Nature Rev. Mol. Cell
Rodriguez-Boulan, E., and Powell, S. K. (1992). Polarity of epithelial
and neuronal cells. Annu. Rev. Cell Biol
Sargiacomo, M., Lisanti, M., Graeve, L., Le Bivic, A., and Rodriguez-
Boulan, E. (1989). Integral and peripheral protein compositions
of the apical and basolateral plasma membrane domains of
MDCK cells. J. Membr. Biol
Yeaman, C., Grindstaff, K.K., and Nelson, W.J. (1999). New perspectives
on mechanisms involved in generating epithelial cell polarity. Physiol. Rev
Zurzolo, C., Le Bivic, A., Quaroni, A., Nitsch, L., and Rodriguez-
Boulan, E. (1992). Modulation of transcytotic and direct targeting
pathways in a polarized thyroid cell line. EMBO J
Zurzolo, C., and Rodriguez-Boulan, E. (1993). Delivery of Na,KATPase
in polarized epithelial cells. Science 260