Protocol

Alkaline Phosphatase Fusion Proteins as Affinity Probes for Protein Localization Studies

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Science's STKE  04 Feb 2003:
Vol. 2003, Issue 168, pp. pl2
DOI: 10.1126/stke.2003.168.pl2

Abstract

The receptor affinity probe or receptor alkaline phosphatase (RAP) staining method uses soluble protein ectodomains fused to secreted placental alkaline phosphatase to locate ectodomain binding sites within cells or tissues. We have used this approach to identify expressing cells in tissue culture, in tissue sections, or in whole-mount embryos. The RAP method is especially useful in situations where a reliable monoclonal antibody is not available or if an orphan receptor is the focus of the study. The technique permits localization of both receptors and ligands and is readily quantifiable for cell-surface binding assays. Soluble ectodomain placental alkaline phosphatase fusion proteins are therefore highly sensitive reagents that permit the direct localization of available binding sites through simple chromogenic assays without purification, radioactive labeling, or secondary reagents.

Introduction

The receptor affinity probe or receptor alkaline phosphatase (RAP) staining method (1-3) uses soluble protein ectodomains fused to secreted placental alkaline phosphatase to locate ectodomain binding sites within cells or tissues. Alkaline phosphatase (AP) has a number of advantages as an indicator protein, including high affinity for a range of substrates, high substrate turnover, and good enzymic stability (4). However, many cells express AP on their surfaces, restricting the usefulness of most AP isoforms as reporter proteins. The RAP assay takes advantage of the fact that, as pointed out by Berger et al. (5), placental AP can be distinguished from other AP isoforms by its resistance to inhibition by l-homoarginine and its relative stability at 65°C. Both of these treatments would effectively eliminate other AP forms. Soluble ectodomain placental AP fusion proteins are therefore highly sensitive reagents that permit the direct localization of available binding sites through simple chromogenic assays without purification, radioactive labeling, or secondary reagents. The approach can be used to indicate ligand-expressing cells if a receptor ectodomain is used, or receptor-expressing cells when a ligand-ectodomain fusion protein is used; it is particularly useful if no antibody is available to the product of the gene of interest, or if an orphan receptor is the focus of the study. We have used this approach to identify expressing cells in tissue culture, in tissue sections, or in whole-mount embryos (3) (Fig. 1). The techniques described will be familiar to any cell biology laboratory that performs antibody-staining procedures. Preparation of the protein reagents requires a minimal understanding of DNA cloning and tissue culture techniques.

Fig 1.

Examples of RAP-stained zebrafish embryos. (A) Cells expressing ephrinB2a following injection of ephrinB2a RNA into the zygote were visualized at 6 hours post fertilization by incubation of the embryo with EphB3-RAP reagent. (B) Anterior view of a 24-hour zebrafish embryo with the eyes removed. Cells expressing ephrinA ligands stain blue following incubation with EphA3RAP reagent (an APtag fusion protein). The tract of the post-optic commissure (white arrowhead) grows along the border of ephrin A-expressing and nonexpressing cells. The black arrowhead indicates the tract of the anterior commissure. (C) Dorsal view of a whole mounted 24-hour embryo stained with ephrinA5AP reagent. Staining indicating that expression of EphA receptors is detected in the temporal half of the eye (arrow), the axons of the anterior commissure (arrowhead), and in rhombomeres 3 and 5 (R3 and R5, respectively). (D) Transverse section through the head of a 48-hour zebrafish embryo. The section has been stained with ephrinA5AP. Staining is detected in the optic nerve, anterior commissure (ac), and retinal ganglion cell layer (gcl) (oc, optic chiasm). (E and F) Whole-mount 48-hour embryos stained with ephrinA5AP. (E) Axons in rhombomeres R3 and R5 of the hindbrain (hb) stain strongly, as do (F) the axons of the retinal ganglion cells innervating the tectal neuropil (tnp). t, tectum; e, eye.

Materials

5-bromo-4-chloro-3-indolyl-phosphate (BCIP)

Acetone

AP Staining Solution (Roche, #1-422-074)

Bovine serum albumin (BSA)

Calcium chloride

Cells for transfection: COS7, COS1, 3T3, or HEK293

Coverslips

Diethanolamine (Sigma-Aldrich, #D8885)

Dulbecco's Modified Eagle's Medium (DMEM) or equivalent, with Glutamax (Autogen Bioclear, #DM12NAB2052)

Fetal calf serum (FCS)

Formaldehyde

Glucose

Glutaraldehyde

Hanks' buffered saline (Sigma-Aldrich, #H9269)

Hepes

l-homoarginine hydrochloride (Sigma-Aldrich, #H1007)

Magnesium chloride

Monoclonal antibody to placental AP (Genzyme) coupled to CNBr-activated sepharose (Genzyme, Cambridge, MA)

N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)

Nitroblue tetrazolium chloride (NBT)

Paraformaldehyde

p-Nitrophenyl Phosphate Liquid Substrate System (Sigma-Aldrich, #N7653)

Penicillin-streptomycin (Sigma-Aldrich, #P3539)

Potassium chloride

Potassium ferricyanide

Potassium ferrocyanide

Potassium hydrogen phosphate (K2HPO4)

Size-exclusion filtration units for use in centrifuges; size cutoff appropriate to fusion protein of interest

Sodium azide (NaN3)

Sodium chloride

Sodium citrate

Sodium hydrogen phosphate (Na2HPO4)

Sterile 1.5-ml microcentrifuge tubes

Sterile 15-ml and 50-ml screw-cap conical centrifuge tubes

Sterile filters (0.2-μm pore) and syringes

Tris base

X-gal

Amplification and Cloning

APtag vectors, APtag-1 and APtag-2 (Available from J. Flanagan, Department of Cell Biology, Harvard Medical School, Boston, MA 02115) (2)

BamHI

BglII

DNA primers designed to amplify the gene of interest, and containing BamHI restriction sites at the ends of both the 5′ and 3′ primers

Qiagen Midiprep or Maxiprep kit (Qiagen)

Reagents for high-fidelity polymerase chain reaction

Equipment

Thermocycler

Transfection

Microcentrifuge tube rack

Multi-speed refrigerated centrifuge

pH probe

Test-tube rack

Tissue culture facilities: Microflow biological safety cabinet, CO2 incubator

Vortexer

Soluble Ectodomain Alkaline Phosphatase Assay (RAP Assay)

96-well flat-bottom culture dish (Corning)

Microtiter plate reader

or Spectrophotometer that can read absorbance at 405 nM

Staining

65°C water bath with means to float slides or sections

Cryostat or freezing microtome (for preparing sections)

Dissection equipment (for preparing tissue for sectioning)

Microscope

PAP Pen for Immunostaining (Sigma-Aldrich, #Z37,782-1)

Recipes

Recipe 1: Medium
FCS10% (w/v)
Penicillin-streptomycin1% (w/v)
Glucose4.5 g/l
Prepare in DMEM. Filter-sterilize. Use at 37°C, but refrigerate when not in use.
Recipe 2: BES-Buffered Saline (BBS)
BES1 M
NaCl1.5 M
Na2HPO41 M
CaCl22.5 M
Adjust the pH to 6.95 with NaOH. Prepare 50 ml in distilled water. Sterile filter. Store at 4°C.
Recipe 3: CaCl2 Solution
Prepare 15 ml of a 2.5-M solution with distilled water. Sterile filter.
Recipe 4: Phosphate-Buffered Saline (PBS)
NaCl137 mM
KCl 2.7 mM
K2HPO41.4 mM
Na2HPO410.1 mM
Adjust the pH to 7.00 with NaOH. Prepare 500 ml in distilled water. Filter sterilize or autoclave and store at 4°C.
Recipe 5: PBS-Based Fixative
Formaldehyde2% (v/w)
Glutaraldehyde0.2% (v/v)
Prepare 15 ml in PBS (Recipe 4). Store at 4°C.
Recipe 6: β-gal Buffer A
Potassium ferricyanide5 mM
Potassium ferrocyanide5 mM
MgCl22 mM
Prepare 50 ml in distilled water. Store at 4°C.
Recipe 7: β-gal Buffer B
Prepare a 10 mM solution of X-gal in β-gal Buffer A (Recipe 6). Prepare 50 ml. Store at 4°C.
Recipe 8: 5× Storage Buffer
Hepes100 mM (pH 7.0)
NaN31%
Prepare 15 ml in distilled water. Store at 4°C.
Recipe 9: Elution Buffer
NaCl 144 mM
MgCl21 mM
CaCl21 mM
Sodium citrate50 mM
Adjust pH to 5.5 with HCl. Prepare 15 ml in distilled water.
Recipe 10: RAP Assay Buffer
Diethanolamine1 M
MgCl20.5 mM
l-homoarginine hydrochloride10 mM
Adjust the pH to 9.8 with NaOH. Prepare 15 ml in distilled water. Store at –20°C.
Recipe 11: ρ-Nitrophenyl Phosphate
Prepare 120 mM ρ-nitrophenyl phosphate in RAP Assay Buffer (Recipe 10). Prepare 15 ml. Store at –20°C.
Recipe 12: Paraformaldehyde (PFA)
Prepare 50 ml of 4% w/v paraformaldehyde in PBS (Recipe 4).
Recipe 13: Hanks' Buffered Saline (Hanks')
NaCl 0.137 M
KCl5.4 mM
Na2HPO40.25 mM
KH2PO40.44 mM
CaCl21.3 mM
MgSO41.0 mM
NaHCO34.2 mM
Prepare 50 ml in distilled water.
Note: Hanks' balanced salt solution (BSA) is also available from Sigma (#H9269).
Recipe 14: Hanks'-BSA
Add 0.5 mg/ml of BSA to Hanks' (Recipe 13). Prepare 50 ml.
Recipe 15: Hepes-Buffered Hanks' Solution (HBHA)
BSA0.5 mg/ml
NaN30.1% (w/v)
Hepes20 mM
Adjust pH to 7.0 with HCl. Prepare 50 ml in Hanks' (Recipe 13).
Recipe 16: Acetone-Formaldehyde Fixative
Acetone60% (v/v)
Formaldehyde3% (v/v)
Hepes20 mM
Adjust the pH to 7.0 with HCl. Prepare 15 ml in distilled water.
Recipe 17: Hepes-Buffered Saline (HBS)
NaCl150 mM
Hepes20 mM
Adjust pH to 7.0 with HCl. Prepare 50 ml in distilled water.
Recipe 18: AP Reaction Buffer
Tris-HCl100 mM
NaCl 100 mM
MgCl250 mM
Adjust pH to 9.5 with HCl. Prepare 15 ml in distilled water.
Recipe 19: AP Staining Buffer
BCIP0.17 mg/ml
NBT 0.33 mg/ml
Prepare in 15 ml in AP Reaction Buffer (Recipe 18).
Note: AP Staining Buffer is also available as a prepared solution from Roche (#1-422-074).

Instructions

Preparation of the Construct and DNA

The cDNA encoding the ectodomain of the gene of interest should be cloned in-frame into the APtag vector of choice (Fig. 1). The vector APtag-1 confers ampicillin resistance to transformed bacterial cells and contains an SV40 origin of replication that selectively enhances the plasmid's production in certain cell lines (1). APtag-1 contains high-level transcription control elements from the Moloney murine leukemia virus long terminal repeat (MoLTR) and rat insulin gene 3′ splice and polyadenylation signals. The cloning site immediately precedes the mature secreted AP protein (5) (Fig. 2C). The KpnI site is at the 3′ end of a 625 base pair (bp) ClaI-KpnI segment of the MoLTR virus, which has recognition sites for SnaBI, EcoRI, SalI, and ClaI at its 5′ end. Cutting the BamHI restriction sites at the ends of the amplification primers for the required DNA sequences permits insertion into the BglII site in APtag-1 to produce a four amino-acid linker to the secreted human placental alkaline phosphatase. APtag-1 is particularly useful for making stable cell lines; for transient transfection, which is much faster and gives, in our hands, similarly high yields, the APtag-2 vector is preferred. The APtag-2 vector contains an immediate early cytomegalovirus (CMV) promoter to drive high-level expression. To obtain these vectors, contact J Flanagan (2).

Fig. 2.

The APtag-1 vector (A) and fusion product (B). SEAP, secreted alkaline phosphatase. The multiple cloning site in the vector is shown in (C).

1. Amplify by high-fidelity polymerase chain reaction (PCR) the coding sequence of the ligand using primers with BamHI restriction sites at the ends of both the 5′ and 3′ primers.

2. Digest the PCR product with Bam HI.

3. Subclone into either the Bg lII-digested APtag-1 (for stable transfections) or APtag-2 (for transient transfections) vectors.

4. Prepare clean, sterile DNA for transfections either by CsCl gradient or using Qiagen Midiprep or Maxiprep kits following the manufacturer's instructions.

Note: Maxiprep DNA prepared by CsCl gradient is ideal; however, DNA prepared using the Qiagen Midiprep or Maxiprep kits also gives good results with transfection rates between 50% and 70%.

Transient Cell Transfection

We have used COS7, COS1, 3T3, or HEK293 cells for production of the AP fusion proteins, depending on availability.

The following stages are performed under sterile conditions.

1. Passage cells and plate at 106 cells per 8-cm tissue culture dish in 10 ml of Medium (Recipe 1).

2. Incubate for 24 hours at 37°C, 5% CO2.

Note: The following volumes are for each petri dish of cells to be transfected.

3. Place 500 μl of BBS (Recipe 2) in a 15-ml screw-cap conical centrifuge tube.

4. Place 20 μg of APtag DNA (encoding the AP fusion protein) containing the subcloned DNA in a microcentrifuge tube and adjust the volume to 450 μl with sterile-filtered distilled water.

5. Add 50 μl of CaCl2 Solution (Recipe 3) to the APtag DNA in the microcentrifuge tube.

6. While vortexing, add the DNA-CaCl2 mixture dropwise to the 15-ml tube containing the BBS (Recipe 2).

7. Wait 15 min, leaving the DNA-CaCl2-BBS mixture at room temperature whilst swirling gently to ensure even distribution.

8. Add all of the solution dropwise to the cells while swirling gently to ensure an even distribution.

9. Incubate for 24 hours at 37°C, 5% CO2.

10. Remove the medium and wash the cells 3 times with 10 ml of sterile PBS (Recipe 4).

11. Replace medium with 10 ml fresh Medium (Recipe 1).

12. Incubate for 3 to 5 days at 37°C, 5% CO2.

13. Perform the RAP Assay to measure the activity of the AP fusion protein in the medium (see below).

Analysis of Transfection Ratio

The following stages are performed under sterile conditions concurrent with the APtag subcloning.

1. Follow the transfection protocol outlined above with a β-galactosidase expression vector.

2. Incubate for 24 hours at 37°C, 5% CO2.

3. Wash the cells 3 times with 10 ml of sterile PBS (Recipe 4).

4. Fix the cells in 2 ml of PBSF (Recipe 5) for 5 min at 4°C.

5. Rinse the cells gently 3 times with 10 ml of PBS (Recipe 4).

6. Rinse the cells in 2 ml β-gal Buffer A (Recipe 6).

7. Stain β-galactosidase-expressing cells by incubating for 2 to 3 hours in 2 ml of β-gal Buffer B (Recipe 7).

8. Assess transfection ratio using light microscopy; β-galactosidase-expressing cells stain blue.

Preparation of Secreted Product (AP Fusion Protein)

The following stages are performed under sterile conditions.

1. Transfer the medium from cells expressing the AP fusion protein to a 15-ml screw-cap conical centrifuge tube.

2. Spin for 1 min at 2000g to pellet cell debris.

3. Assay medium for RAP activity (see below) to determine concentration of the AP fusion protein. Concentrate if necessary (see below).

Note: Concentration can also be assayed by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining.

4. Adjust concentration of AP to about 25 mM with 0.25 volumes of 5× Storage Buffer (Recipe 8) for long-term storage, and store at 4°C.

Concentration by size exclusion filtration

1. Transfer supernatant to appropriate size exclusion filters; 30 kD is often suitable due to the size of RAP.

2. Spin for 1 hour at 2000g at 4°C.

Note: Depending on the size of the secreted protein and the exclusion filters used, spin times may need to be adjusted.

3. Transfer retained material to an appropriate container, such as 1.5-ml centrifuge tubes.

4. Adjust solution to required concentration with 5× Storage Buffer (Recipe 8). Store at 4°C.

Note: The APtag fusion proteins appear to be stable when stored in tissue culture supernatant at 4°C for several months. Indeed, specific activity has been observed after 4 years in some cases.

Concentration by antibody precipitation

Concentration and purification of protein can be achieved by incubation with monoclonal antibody to placental AP (Genzyme) coupled to CNBr-activated sepharose.

1. Incubate the culture medium from the APtag-transfected cells with an appropriate volume of monoclonal antibody to placental AP.

2. Elute the bound protein with an appropriate volume of Elution Buffer (Recipe 9) and immediately neutralize with 1 M Hepes (pH 8.0).

Note: For staining purposes, purification of the APtag fusion protein in this manner is not necessary.

RAP Assay

1. Add a 10-μl aliquot of medium to 190 μl RAP Assay Buffer (Recipe 10).

Note: Heating the samples to 65°C for 10 min and including 10 mM l-homoarginine inhibits any endogenous phosphatase activity.

2. Incubate at 37°C for 10 min in a 96-well flat-bottom culture dish.

Note: Performing the assay at 37°C doubles the reaction rate, but also increases the rate of spontaneous chemical hydrolysis of substrate. The reaction can also be performed at room temperature if necessary.

3. Add 20 μl of pre-warmed (37°C) ρ-Nitrophenyl Phosphate (Recipe 11) and mix well.

4. Read absorbance at 405 nm (A405) of mixture in an Artek automatic plate reader (or equivalent) at room temperature at 1-min intervals.

5. Calculate the maximum linear reaction rate. A change of 30 OD units/hour corresponds to about 1 pmol of AP fusion protein under the assay conditions.

Note: Specific activity for RAP is estimated at 2000 U/mg (5).

Staining of Whole-Mount Embryos

We use zebrafish embryos, but the protocol should work well in other species as long as adjustments are made for the size of the tissue. In general, procedures that work well for antibody staining work well for RAP staining.

1. Fix the tissue in 100 μl (or an appropriate volume) of PFA (Recipe 12) for 1 to 2 hours at room temperature.

Note: For embryos 48 hours and older, methods to increase penetration may be necessary. For example, incubation of embryos in methanol overnight at –20°C after fixation and before staining may be helpful. As with antibody staining, in some instances incubation in methanol may affect the ability of the reagent to bind. An alternative method to increase penetration is to briefly incubate the embryos in chilled trypsin. 0.25% trypsin is one possible concentration, but the required amount should be empirically determined for each batch of trypsin.

2. Wash the fixed embryos four times for 5 min in 100 μl (or an appropriate volume) of Hanks'-BSA (Recipe 14).

3. Incubate in AP fusion protein (diluted as appropriate in distilled water or HBS) for 90 min.

Note: Depending on the tissue under study, rotation during the incubation may improve staining.

Note: Concentrations of AP fusion protein up to 1 μM have been used successfully when staining whole-mount zebrafish embryos. The required concentration will depend on the binding affinity of the receptor-ligand reaction.

4. Wash six times for 5 min in 100 μl (or an appropriate volume) of HBHA (Recipe 15)

5. Incubate in 100 μl (or an appropriate volume) of Acetone-Formaldehyde Fixative (Recipe 16) for 2.5 min.

6. Wash three times for 5 min in 100 μl (or an appropriate volume) of HBS (Recipe 17).

7. Incubate for 20 to 30 min at 65 to 70°C to kill endogenous alkaline phosphatase.

8. Wash once in 100 μl (or an appropriate volume) of AP Reaction Buffer (Recipe 18).

9. Incubate in AP Staining Buffer (Recipe 19) until required level of staining is achieved.

Note: Staining Buffer is light sensitive, so incubation should be performed in the dark.

Note: The staining can be performed at room temperature or at 37°C; however, performing the assay at 37°C doubles the reaction rate and also increases the rate of spontaneous chemical hydrolysis of substrate.

Staining of Tissue Sections

These methods have been previously described (6). We recommend sectioning directly onto slides and using a PAP pen, which will permit rapid washing and conservation of reagents.

1. Snap-freeze dissected tissue and immediately section (20 to 40 μm thick) and process for RAP binding.

Note: Tissue that has been fixed before sectioning can also be used. Fix for 2 hours at room temperature in PFA (Recipe 12). Sucrose fixation may be used as an alternative.

2. Mark a circle around the sections with a PAP pen.

3. Rehydrate the sections for 10 min in 100 μl (or an appropriate volume) of HBHA (Recipe 15).

4. Incubate for 2 hours in the appropriate AP fusion protein at 7 nM, diluted in HBS.

Note: Testing various concentrations of the AP fusion protein may be necessary. For EphR-APtag, staining concentrations of 3 to 10 nM have been used when staining sections (6).

5. Rinse four times in an appropriate volume of HBS (Recipe 17).

6. Fix for 90 s in an appropriate volume of Acetone-Formaldehyde Fixative (Recipe 16).

7. Wash six times in an appropriate volume of HBS (Recipe 17).

8. Incubate at 65°C for 1 hour to reduce endogenous AP activity.

9. Wash in an appropriate volume of AP Reaction Buffer (Recipe 18) for 10 min.

Note: Inclusion of 10 mM homoarginine may improve the quality of staining by reducing background AP activity.

10. Incubate in AP Staining Buffer (Recipe 19) until required color intensity has been reached.

References

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