Apr 07, 2025

Public workspaceEffective polyclonal antibodies against the virulence-associated protein D (vapD) of Helicobacter pylori, obtained from recombinant VapD

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.dm6gpz251lzp/v1
  • Alejandro Flores-Alanis1,
  • Gabriela Delgado1,
  • Carlos Santiago-Olivares1,
  • Víctor Manuel Luna-Pineda2,
  • Armando Cruz-Rangel3,
  • Denilson Guerrero-Mejía4,
  • María Luisa Escobar-Sánchez5,
  • Nayeli Torres-Ramírez5,
  • Rosario Morales-Espinosa1
  • 1Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico;
  • 2Laboratorio de Investigación en Patógenos Respiratorios y Producción de Biológicos, Hospital Infantil de México “Federico Gómez”, Mexico City, Mexico;
  • 3Laboratorio de Bioquímica de Enfermedades Crónicas, Instituto Nacional de Medicina Genómica, Mexico City, Mexico;
  • 4Laboratorio de Inmunobiología y Diagnóstico Molecular, Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Guerrero, Mexico;
  • 5Departamento de Biología Celular, Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico City, Mexico
  • PLOS ONE Lab Protocols
    Tech. support email: plosone@plos.org
Alejandro Flores-Alanis
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpz251lzp/v1
External link: https://doi.org/10.1371/journal.pone.0321455
Protocol Citation: Alejandro Flores-Alanis, Gabriela Delgado, Carlos Santiago-Olivares, Víctor Manuel Luna-Pineda, Armando Cruz-Rangel, Denilson Guerrero-Mejía, María Luisa Escobar-Sánchez, Nayeli Torres-Ramírez, Rosario Morales-Espinosa 2025. Effective polyclonal antibodies against the virulence-associated protein D (vapD) of Helicobacter pylori, obtained from recombinant VapD. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpz251lzp/v1
Manuscript citation:
Flores-Alanis A, Delgado G, Santiago-Olivares C, Luna-Pineda VM, Cruz-Rangel A, Guerrero-Mejía D, Escobar-Sánchez ML, Torres-Ramírez N, Morales-Espinosa R (2025) Effective polyclonal antibodies against the virulence-associated protein D (VapD) of Helicobacter pylori, obtained from recombinant VapD. PLOS One 20(4). doi: 10.1371/journal.pone.0321455
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 10, 2024
Last Modified: April 07, 2025
Protocol Integer ID: 105161
Keywords: Helicobacter pylori, virulence-associated protein D (VapD), recombinant protein, protein purification, polyclonal antibodies
Funders Acknowledgements:
DGAPA-PAPIIT
Grant ID: IN213921
CONAHCYT
Grant ID: CF-2023-G-919
Abstract
Helicobacter pylori is a microorganism associated with serious gastric pathologies. This bacterium presents specific genes that encode for different virulence factors associated with the development of gastric disease. The VapD protein has rarely been studied, although it has been previously demonstrated its participation in the protection of Helicobacter pylori within gastric cells. In the present work, we document the protocols developed to generate the VapD recombinant protein and the subsequent production of polyclonal antibodies. Our research group faced several problems throughout the trials; however, all of them were successfully solved.
Materials
Materials and reagents:

  1. Synthetic primers for PCR amplification
  2. Agarose gels
  3. LB medium
  4. LB agar plates containing ampicillin (100 µg/ml)
  5. Blood agar plates
  6. Electroporation cuvettes of 0.1 cm
  7. 15 ml tube with a screw cap
  8. 250 ml Erlenmeyer flask
  9. Isopropyl-thio-β-D-galactopyranoside (IPTG)
  10. EDTA-free protease inhibitor cocktail
  11. Phenylmethylsulfonyl fluoride (PMSF)
  12. 20% SDS-PAGE gels
  13. Chromatography column
  14. Skimmed milk powder
  15. 96-well high binding plate
  16. Nitrocellulose membrane
  17. PVDF membrane
  18. Dulbecco´s Modified Eagle's Medium (DMEM)
  19. Fetal bovine serum
  20. 60 mm crystal polystyrene Petri dishes
  21. 120 mm crystal polystyrene Petri dishes
  22. Coverslips
  23. HisPur™ Ni-NTA Resin (Thermo Fisher Scientific, Massachusetts, USA)
  24. HiPrep 26/10 Desalting (Cytiva™, Massachusetts, USA)
  25. Freund’s incomplete adjuvant (Sigma-Aldrich, Massachusetts, USA)
  26. Chemiluminescence reagent SuperSignal™ West Pico PLUS (Thermo Fisher Scientific, Massachusetts)
  27. Antifade Mounting Medium (Vectashield®, California, USA)
  28. Quick Start™ Bradford 1X dye reagent (Bio-Rad, California, USA)
  29. Protein A/G agarose beads (Santa Cruz Biotechnology Inc. California, USA)
  30. Coomassie® Brilliant blue R 250 (Merck, New Jersey, USA)

Kits:

  1. ReagentPlatinum™ Taq DNA Polymerase High FidelityThermo FisherCatalog #11304011 .
  2. ReagentMinElute Gel extraction kitQiagenCatalog #28604 .
  3. ReagentaLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag)Thermo FisherCatalog #K1261 .
  4. ReagentPureDireX, Plasmid miniPREP KitBio-HelixCatalog #PDP01-0100 .

Bacterial strains and cell culture:

  1. Competent Escherichia coli DH5α
  2. Competent Escherichia coli Rosetta (DE3)
  3. Helicobacter pylori strain 26695 (ATCC 700392)
  4. Adenocarcinoma cell line (AGS) (ATCC CRL-1739)

Antibodies:
  1. Antibody anti-mouse H+L coupled to horseradish peroxidase (Santa Cruz Biotechnology Inc. California, USA)
  2. Antibody anti-H. pylori developed in rabbit (Biocare Medical, California, USA)
  3. Antibody anti-mouse Alexa Fluor 594 (Life Technologies, Eugene, OR)
  4. Antibody anti-rabbit Alexa Fluor 488 (Life Technologies, Eugene, OR)

Buffers and solutions:
  • TE buffer
AB
Tris HCl, pH 8.010 mM
EDTA1 mM
  • BMR4× buffer
AB
Tris, pH 6.8320 mM
SDS8 % w/v
Glycerol40% v/v
Bromophenol blue0.008 % w/v
2-Mercaptoethanol 21 % v/v
  • SDS-PAGE running buffer 10×
AB
Tris 250 mM
Glycine2.5 M
SDS1 % w/v
  • Coomassie brilliant blue solution
AB
Coomassie brilliant blue R-2500.5 % w/v
Methanol50 % v/v
acetic acid7 % v/v
  • Lysis buffer
AB
Tris HCl, pH 8.050 mM
Glycerol5 % v/v
2-Mercaptoethanol 1 mM
EDTA0.1 mM
  • Equilibration buffer
AB
Tris HCl, pH 8.050 mM
NaCl150 mM
EDTA0.1 mM
DTT1 mM
  • Wash buffer (equilibration buffer containing 50 mM imidazole)
  • Elution buffer (equilibration buffer containing 325 mM imidazole)
  • PBS 10×
AB
NaCl1.37 M
KCl27 mM
Na2HPO4100 mM
KH2PO418 mM
pH 7.4
  • Carbonate-bicarbonate buffer
AB
Na2CO34 mM
NaHCO346 mM
pH9.2
  • PBT (PBS 1× containing 0.1% v/v Tween 20)
  • Phosphate-citrate buffer
AB
Na2HPO451 mM
Citric acid [HOC(CH₂CO₂H)₂] 24 mM
  • OPD developer solution
AB
Phosphate-citrate buffer, pH 5.050 mM
Ortho-102 phenylenediamine (OPD) 0.4 mg/ml
Hydrogen peroxide (H2O2) 30% v/v
Add the H2O2 immediately prior the use
  • DAB developer solution (PBS 1× containing 0.5 mg/ml of 3,3′-diaminobenzidine (DAB) and 30% v/v H2O2). Make the solution just before use.
  • Western blot transfer buffer (SDS-PAGE running buffer 1× 10% v/v, methanol 20% v/v)
  • Fixative solution (PBS 1× pH 7.2, 2% v/v paraformaldehyde)
  • Permeabilizing solution (PBS 1× pH 7.2, Triton X-100 0.5% v/v)

Equipment:
  1. MJ Mini™ 113 PCR thermal cycler (Bio-Rad, California, USA)
  2. Spectrophotometer NanoDrop™ 114 (Thermo Fisher Scientific, Massachusetts, USA).
  3. Horizontal electrophoresis chamber (Bio-Rad, California, USA. No cat.1704482).
  4. Ultra-violet (UV) transilluminator (Analytikjena, Jena, Germany)
  5. Electroporator Gene Pulser Xcell Microbial system (Bio-Rad, California, USA. No cat.1652666)
  6. Incubator at 37 ºC (Thermo Fisher Scientific, Massachusetts, USA)
  7. Shaker incubator at 37 ºC (mrc Laboratory Instruments, Harlow, UK)
  8. Microplate reader spectrophotometer (TECAN, Männedorf, Switzerland)
  9. SDS-PAGE electrophoresis chamber (Bio-Rad, California, USA. No cat.1652666)
  10. Sonic dismembrator Fisherbrand™ 126 (Thermo Fisher Scientific, Massachusetts, USA)
  11. Refrigerated ultracentrifuge (Thermo Fisher Scientific, Massachusetts, USA)
  12. FPLC (Cytiva™ 129 , Massachusetts, USA)
  13. Tank blotting system (Bio-Rad, California, USA)
  14. C-DiGit® 131 blot scanner (LI-COR, Nebraska, USA)
  15. Nikon Eclipse E600 epifluorescence microscope (Nikon, NY, USA)

Before start
The reagents, vendors and manufacturers used can be replaced by similar ones.
Construction of the expression plasmid and bacteria transformation
Construction of the expression plasmid and bacteria transformation
8h 28m
8h 28m
The vapD gene was amplified by PCR. The PCR mixture was prepared as follows:
AB
High fidelity buffer1X
MgSO41.5 mM
dNTPs0.6 mM
Primer VapDcter forward 5’-AGA AGG AGA TAT AAC TAT GTA TGC GCT GGC GTT TGA TCT-3’ 0.5 µM
Primer VapDcter reverse 5’-GTG GTG GTG ATG GTG ATG GCC GCT TTT CAC AAT TTC GGT-3’0.5 µM
Platinum ® 140 Taq DNA polymerase High Fidelity1U
DNA (the plasmid construction pCR 2.1-vapD was used as template) 1 µl
Water for final volume of 25 µlX µl


Pipetting
Mix
Program the PCR thermal cycler with the following conditions: denaturation at Temperature94 °C for Duration00:05:00 , followed by 30 cycles of denaturation at Temperature94 °C for Duration00:01:00 , annealing at Temperature52 °C for Duration00:01:00 and extension at Temperature72 °C for Duration00:01:00 ; and a final extension at Temperature72 °C for Duration00:10:00 .

18m
PCR
Run the PCR product by electrophoresis on agarose gel and visualize it on an UV transilluminator.

Analyze
Purify the PCR product from agarose gel electrophoresis using the MinElute Gel Extraction Kit following the manufacturer's instructions.

Quantify the PCR product by measuring its absorbance at 260/280 nm using a spectrophotometer.
Analyze
Generate the VapD-6xHis fusion vector using the aLICator™ LIC Cloning and Expression System Kit 3:

Add Amount2 µL of 5× LIC buffer, Amount1 µL of T4 DNA polymerase, Amount20 ng of purified PCR product, and enough water to adjust the final volume to Amount9 µL . Vortex briefly.

Pipetting
Mix
Incubate the reaction mixture at TemperatureRoom temperature for Duration00:05:00 .

5m
Incubation
Stop the reaction by adding Amount0.6 µL of Concentration0.5 Molarity (M) EDTA and mix well.

Pipetting
Mix
For annealing, add Amount1 µL of pLATE31 LIC-ready vector (Amount60 ng ) to the reaction. Vortex briefly.

Pipetting
Incubate the annealing reaction at TemperatureRoom temperature for Duration00:05:00 .
Note
We attempted to clone vapD gene into various expression vectors, including pGEx-4T-2, pET-28b(+), and pET30-GBFusion1, but were not successful.


5m
Incubation
Transform by electroporation Amount1 µL -Amount6 µL of the annealing reaction into Amount50 µL of competent E. coli DH5α cells. Deliver the electric pulse under the following conditions: 1.8 kV, 200 mA for 0.5 milisecs.

Note
If an electroporator is unavailable, thermal shock can be used for transformation.

Pipetting
Spread Amount50 µL of the cells on an LB agar plate containing ampicillin (Amount100 µg /mL ).
Note
If no colonies appear after transformation, centrifuge the bacterial culture at 500 xg, discard the supernatant, and add 100 µl of fresh medium. Resuspend the cell pellet thoroughly and spread it onto a plate.     


Incubate at Temperature37 °C Overnight .

8h
Incubation
Overnight
Selection of positive E. coli DH5α colonies
Selection of positive E. coli DH5α colonies
8h
8h
Pick, at least 10 colonies and perform colony PCR screening using the same conditions to amplify the vapD gene (see steps 1 and 2).

PCR
Inoculate Amount5 mL of LB medium containing ampicillin (Amount100 µg /mL ) with the positive colonies.

Incubate the tubes at Temperature37 °C Overnight in a shaking incubator at Shaker200 rpm /min .

8h
Incubation
Overnight
Plasmid extraction and verification of the expression vector (pLATE31-VapD-6xHis)
Plasmid extraction and verification of the expression vector (pLATE31-VapD-6xHis)
Extract the plasmid from saturated cultures using the PureDirex Plasmid miniPREP Kit, following the manufacturer’s instructions.

Run the plasmids by electrophoresis on agarose gel.
Visualize the gel on an UV transilluminator.

Analyze
Screened the positives plasmids for the vapD gene by Sanger sequencing to ensure that there are not PCR-introduced mutations, using the LIC forward and LIC reverse sequencing primers included in the aLICator™ Ligation Independent Cloning and Expression System Kit 3.

Analyze
Computational step
Transformation of E. coli Rosetta (DE3)
Transformation of E. coli Rosetta (DE3)
8h
8h
Transform by electroporation competent E. coli Rosetta (DE3) cells with Amount10 ng of the expression vector (pLATE31-VapD-6xHis). Deliver the electric pulse under the following conditions: 1.8 kV, 200 mA for 0.5milisecs.

Note
If an electroporator is unavailable, thermal shock can be used for transformation. 
We attempted to clone vapD gene into BL21 (DE3), but the attempted was unsuccessful.

Pipetting
Spread Amount50 µL of the cells on an LB agar plate containing ampicillin (Amount100 µg /mL ).
Note
If no colonies appear after transformation, centrifuge the bacterial culture at 500 xg, discard the supernatant, and add 100 µl of fresh medium. Resuspend the cell pellet and spread thoroughly it onto the plate.     


Incubate the plates at Temperature37 °C Overnight .

8h
Incubation
Overnight
Expression of the recombinant protein part 1
Expression of the recombinant protein part 1
8h
8h
Inoculate a 15 ml tube with a screw tap with Amount3 mL of LB medium containing ampicillin(Amount100 µg /mL ) with a single colony of E. coli Rosetta (DE3) containing the expression vector (pLATE31-VapD-6xHis).

Incubate the tube at Temperature37 °C Overnight in a shaking incubator at Shaker200 rpm /min .

8h
Incubation
Overnight
Expression of the recombinant protein part 2
Expression of the recombinant protein part 2
4h
4h
Inoculate Amount30 mL of LB medium in a 250 ml Erlenmeyer flask with Amount0.5 mL of saturated overnight culture.

Pipetting
Grow the cells at Temperature37 °C in a shaking incubator at Shaker200 rpm /min to mild-log phase (OD600nm ca. 0.5).

Incubation
Add Concentration0.1 millimolar (mM) final concentration of IPTG.

Pipetting
Incubate the culture for Duration04:00:00 at Temperature37 °C with shaking.

4h
Incubation
Transfer the culture to a 50 ml conical tube and centrifuge at Centrifigation3000 rpm, 4°C , discard the supernatant. The cell pellet can be stored at Temperature-70 °C until use.

Note
We tested different concentrations of IPT (0.1, 0.5, and 1.0 mM) and incubation times, including overnight expression, but observed no differences. The optimal conditions were as previously mentioned.
To verify the correct expression of the recombinant vapD protein (rVapD), take Amount20 µL of the culture before centrifugation, add Amount2.5 µL of 4× BMR buffer, and boil it for Duration00:03:00 . Separate the sample using SDS-PAGE and visualize it with Coomassie brilliant blue staining.

Centrifigation
Purification of rVapD by Immobilized Metal Affinity Chromatography (IMAC)
Purification of rVapD by Immobilized Metal Affinity Chromatography (IMAC)
48m 20s
48m 20s
Resuspend the cell pellet in Amount10 mL of lysis buffer supplemented with EDTA-free protease inhibitor cocktail and PMSF Concentration0.2 millimolar (mM) .

Add 0.7% sarkosyl, 3% Triton X-100, and Concentration20 millimolar (mM) CHAPS, then mix gently for Duration00:15:00 at Temperature4 °C using an end-over-end rotator.

15m
Lyse the cells by sonication, pulses of Duration00:00:10 at 70% of amplitude with intervals of Duration00:00:10 between each pulse, up to reach a clear cell lysate (ca. Duration00:10:00 ).

Note
If a sonic dismembrator is unavailable, the cell lysis can be performed using lysozyme as follows:
  • Add Amount1 mg /mL of lysozyme and incubate forDuration01:00:00 at Temperature4 °C with gentle agitation at end-over-end rotator.
  • Add 0.7% sarkosyl, 3% Triton X-100, and Concentration20 millimolar (mM) CHAPS, then mix gently for Duration00:15:00 at Temperature4 °C using an end-over-end rotator.

10m 20s
Centrifuge at Centrifigation15000 x g, 00:20:00 and recover the supernatant in conical tubes of 50 ml.

Note
Sarkosyl can be used at higher concentrations (1-10%) to maximize the recovery of insoluble protein. However, we observed that the addition of sarkosyl (concentration >1%) increased the viscosity of the protein solution, with higher concentrations leading to greater viscosity. If the protein solution becomes too viscous, it can be passed through sterile gauze and gently squeezed, stopping when the filtered solution begins to thicken again.

20m
Centrifigation
Add an appropriate amount of Ni-NTA resin to a chromatography column. Once the column is packed, equilibrate it with two column volumes of equilibration buffer.

Add the protein solution onto the column at a flow rate of Amount1 mL /min .

Wash the column with five column volumes of wash buffer.

Wash
Elute the rVapD protein from the resin with five volumes of elution buffer at a flow rate of Amount1 mL /min .

Pass the fractions containing the rVapD through a HiPrep 26/10 Desalting at a flow rate of Amount1 mL /min to remove imidazole.

Note
If a desalting column is unavailable, the imidazole can be removed by dialysis.
Analyze the purified rVapD. Take Amount20 µL of the sample, add Amount2.5 µL of 4× BMR buffer, and boil it for Duration00:03:00 .

3m
Pipetting
Analyze
Separate the sample by SDS-PAGE.

Stain the gel with Coomassie brilliant blue solution.

Production of polyclonal antibodies against VapD
Production of polyclonal antibodies against VapD
A total of six six-week-old Balb/C mice were used. The mice were intraperitoneally immunized five times, fortnightly, with Amount10 µg of rVapD diluted in PBS 1× and mixed with Freund’s incomplete adjuvant, in a total volume of Amount200 µL per animal. Four serum samples were collected. After the final sample collection, the mice were euthanized with CO2. The experiment is depicted in Fig 1.

Fig 1. Representation of the immunization schedule. Sera were collected for antibody analysis at specific time point: before the first inoculation (pre-immune serum) and at 15, 45, and 60 days after each booster doce (gray arrows). The final blood collection was performed on day 75 (red arrow). This figure was created with https://www.biorender.com/

Titration of polyclonal serum by indirect ELISA and Dot Blot
Titration of polyclonal serum by indirect ELISA and Dot Blot
1d 1h 10m
1d 1h 10m
Dilute rVapD in carbonate-bicarbonate buffer a final concentration of Amount100 ng /mL .
Coat a flat-bottomed 96-well high binding plate with Amount100 µL /well of rVapD diluted in carbonate buffer.

Pipetting
Incubate DurationOvernight at Temperature4 °C .
8h
Incubation
Overnight
Wash three times Amount200 µL /well of PBT.

Wash
Block the unoccupied sites with Amount200 µL /well of 5% skimmed milk diluted in PBT.

Pipetting
Incubate for Duration00:40:00 at Temperature37 °C .

40m
Incubation
Incubate the plates for Duration02:00:00 at Temperature37 °C with Amount100 µL /well of serum from immunized mice, prepared in serial two-fold dilutions ranging from 1:500 to 1:32,000.

2h
Incubation
Pipetting
Wash three times with Amount200 µL /well of PBT.

Wash
Incubate the plate with the secondary antibody mouse antibody H+L coupled to horseradish peroxidase for Duration01:30:00 at Temperature37 °C .

1h 30m
Incubation
Wash three times with Amount200 µL /well of PBT.

Wash
Add Amount100 µL /well of the OPD developer solution.

Pipetting
Incubate for Duration00:20:00 at TemperatureRoom temperature .
20m
Incubation
Stop the reaction with Amount100 µL /well of Concentration3 Molarity (M) sulfuric acid.

Pipetting
Read at 492nm in a spectrophotometer.
Analyze
For the dot blot, fix a nitrocellulose membrane with different concentrations of rVapD DurationOvernight at Temperature4 °C . The initial concentration is Amount1 µg /mL of rVapD, serially diluted two-fold down to Amount0.03 µg /mL .

8h
Pipetting
Overnight
Block the membranes with 5% skimmed milk diluted in PBT and incubate for Duration00:40:00 with gentle shaking at TemperatureRoom temperature .

40m
Incubation
Wash the membrane three times with PBT.

Wash
Incubate with the mouse serum (anti-VapD) at dilution of 1:500, 1:1,000, and 1:2,000 for Duration02:00:00 with gentle shaking at TemperatureRoom temperature .

2h
Incubation
Wash the membrane three times with PBT.

Wash
Incubate with the secondary antibody (anti-mouse antibody H+L coupled to horseradish peroxidase) at a 1:2,000 dilution for Duration02:00:00 with gentle shaking at TemperatureRoom temperature .

2h
Incubation
Visualize the enzymatic reaction with the DAB developer solution and stop it with distilled water.

Analyze
Western blot anti-VapD
Western blot anti-VapD
11h 20m
11h 20m
Load in 20% SDS-PAGE gels with different concentrations of rVapD, and run.

Transfer the SDS-PAGE gel to a PVDF membrane for Duration01:00:00 at a constant voltage of 100 V at Temperature4 °C .

1h
Block the unoccupied sites with 3% skimmed milk in PBS 1× for Duration01:00:00 at TemperatureRoom temperature with gentle rotation.

1h
Incubate the membrane with mouse serum (anti-VapD) as the primary antibody in a dilution of 1:2,000 DurationOvernight at Temperature4 °C with gentle agitation.

8h
Incubation
Wash three times with PBT for Duration00:10:00 .

10m
Wash
Incubate the membrane with the secondary antibody (anti-mouse antibody H+L coupled to horseradish peroxidase) at a 1:2,000 dilution forDuration01:00:00 at Temperature4 °C with gentle agitation.

1h
Incubation
Wash three times with PBT for Duration00:10:00 .

10m
Wash
Perform the protein detection using a chemiluminescence reagent and a blot scanner.

Note
If a blot scanner is unavailable, the chemiluminescence signal can be detected in a dark room using radiographic plaques.

Analyze
Detection of VapD in Helicobacter pylori and AGS cells co-cultures
Detection of VapD in Helicobacter pylori and AGS cells co-cultures
4d 23h 26m
4d 23h 26m
Grow Human gastric adenocarcinoma cell lines (AGS) in DMEM supplemented with 5% fetal bovine serum, 100 IU/mL penicillin, Amount100 mg /mL streptomycin, 0.2% NaHCO3, Concentration10 millimolar (mM) HEPES, and Concentration2 millimolar (mM) glutamine.

Incubate at Temperature37 °C in humidified atmosphere, in the presence of 5% CO2.

Incubation
Grow H. pylori strain 26695 in blood agar plates supplemented with 5% fetal bovine serum.

Incubate at Temperature37 °C for Duration48:00:00 in humidified atmosphere, in the presence of 8% CO2.

2d
Incubation
Seed 2.5x105 AGS cells in 35 mm Petri dishes.

Incubate at Temperature37 °C DurationOvernight in humidified atmosphere, in the presence of 5% CO2.

8h
Incubation
Wash three times with PBS 1× and inoculate H. pylori strain 26695 at MOI of 100 (multiplicity of infection of 100 bacteria/cell) in Amount2.5 mL of DMEM.

Wash
Incubate at Temperature37 °C for Duration03:00:00 in humidified atmosphere, in the presence of 5% CO2.

3h
Incubation
Remove the supernatant, and add Amount2.5 mL of DMEM containing Amount200 µg /mL gentamicin.

Pipetting
Incubate for an additional Duration02:00:00 .

2h
Incubation
Wash three times with PBS 1×.

Wash
Add Amount2.5 mL of DMEM supplemented with 2% fetal bovine serum.
Pipetting
Place three coverslips in each Petri dish and incubate at Temperature37 °C in a humidified atmosphere in the presence of 5% CO2 for up to Duration48:00:00 .

2d
Incubation
Wash the cells with PBS 1×.

Wash
Fix with fixative solution for Duration00:20:00 at TemperatureRoom temperature .
20m
Wash the cells with PBS 1× and permeabilize them with permeabilizing solution for Duration00:05:00 at Temperature4 °C .

5m
Wash
Incubate the cells with mouse serum (anti- vapD) at a dilution of 1:100 in PBS 1× and anti-H. pylori at a dilution of 1:200, DurationOvernight at Temperature4 °C .
8h
Incubation
Overnight
Wash the cells with PBS 1×.

Wash
Incubate for Duration02:00:00 at TemperatureRoom temperature with anti-mouse Alexa Fluor 594 and anti-rabbit Alexa Fluor 488 antibodies diluted 1:100 in PBS 1×.

2h
Incubation
Wash the cells with PBS 1× .

Wash
Incubate with DAPI (4´, 6-diamino-2-phenilindol) for Duration00:01:00 at TemperatureRoom temperature .

1m
Incubation
Wash the cells with PBS 1X.
Wash
Mount with mounting medium for fluorescence (Antifade Mounting Medium).
Observe the samples under an epifluorescence microscope.

Imaging
Immunoprecipitation of VapD
Immunoprecipitation of VapD
4h 5m
4h 5m
Wash the protein A/G agarose beads (Amount20 µL per reaction) two times with Amount200 µL of lysis buffer supplemented with EDTA-free protease inhibitor cocktail and Concentration0.2 millimolar (mM) PMSF.

Wash
Centrifuge at Centrifigation500 x g, 00:01:00 .

1m
Centrifigation
Resuspend the washed beads in five volumes of lysis buffer.
Pipetting
Incubate the beads with Amount5 µL of mouse serum (anti-VapD) diluted in lysis buffer for Duration02:00:00 atTemperature4 °C with gentle agitation.

2h
Incubation
Recover the beads by centrifugation at Centrifigation500 x g, 4°C, 00:01:00 , and discarding the supernatant (unbound antibody).
1m
Centrifigation
Wash the beads two times with 10 volumes of lysis buffer and maintain them atTemperature4 °C .

Wash
Dilute Amount20 µL of lysate of Rosetta (DE3) overexpressing rVapD with lysis buffer to a final volume of Amount100 µL .

Pipetting
Incubate the antibody-coupled beads with the bacterial lysate for Duration02:00:00 at Temperature4 °C with gentle agitation.

2h
Incubation
Collect the immunoprecipitates by centrifugation at Centrifigation500 x g, 4°C .

Centrifigation
Wash three times with Amount100 µL of lysis buffer.

Wash
Elute the proteins complexes by adding Amount20 µL of 4× BMR buffer and boiling for Duration00:03:00 .

3m
Load the samples on 20% SDS-PAGE gels and run.
Stain the gel with Coomassie brilliant blue solution.
Acknowledgements
We thank Jose Luis Mendez for helpful laboratory techniques.