Apr 21, 2023
Version 1
CagA phosphorylation assay and its semiquantitative analysis V.1
- Anna F. Zeitler1,
- Luisa F Jimenez-Soto2
- 1Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, Pettenkoferstraße 9a, D-80336 Munich, Germany;
- 2Walther Straub Institute for Pharmacology and Toxicology
Protocol Citation: Anna F. Zeitler, Luisa F Jimenez-Soto 2023. CagA phosphorylation assay and its semiquantitative analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8x6l5r7/v1
Manuscript citation:
Jiménez-Soto, L. F. and Haas, R. The CagA toxin of Helicobacter pylori: abundant production but relatively low amount translocated.Sci. Rep.6, 23227; doi: 10.1038/srep23227 (2016).
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 is working.
Created: December 07, 2016
Last Modified: April 21, 2023
Protocol Integer ID: 4549
Keywords: Helicobacter pylori, CagA phosphorylation, cellular infection
Disclaimer
These protocol is the result of transfer of information across several generation of PhD students under the guidance of Prof. Rainer Haas and PD. Dr. Wolfgang Fischer. The steps here are the authors' finished polished product of protocols already existent in the laboratory.
Abstract
CagA is the oncogenic toxic of Helicobacter pylori. The toxin is injected into host cells and tyrosine phosphorylated in the C-terminal EPIYA motifs by host kinases. Positive translocation of the CagA toxin into the cell is evaluated by detecting the phosphorylated form in the cell lysate using antibodies detecting phosphotyrosines. Previously detection was limited to a positive o negative results, but with new advances in semiquantitative western blots we are able to quantify changes on CagA translocation / Phosphorylation and get a better idea of the dynamics of injection.
The protocol described here has been used for evaluation of CagA phosphorylation and its semiquantitative analysis in the following publications:
Guidelines
Make sure you follow the biosafety protocols and recommendations of your laboratory.
Materials
OXOID GC AGAR BASE (CM0367GC) Media was used. Its preparation follows the indications of the manufacturer.
For the preparation of the vitamin mix stock solution (100X), contact directly Prof. Rainer Haas or PD. Dr. Wolfgang Fischer at the Max-von-Pettenkofer Institute in Munich, Germany.
Safety warnings
Components like Methanol and Acrylamide are know to cause damage to the nervous system. Work with them under the chemical hood to protect you from inhaling their vapors.
Ethics statement
These experiments do not need or include the use of animals or human material.
Before start
Make sure you know how to use all equipment in the lab, and read the safety warning of all chemicals used before you use them.
Prepare H. pylori bacteria for infection
Prepare H. pylori bacteria for infection
H. pylori has a amazing capacity to change during growth. In order to keep the experiments reproducible and comparable, use relative fresh bacteria for all infection experiments.
On Friday afternoon, take from frozen stocks the bacteria by scratching the surface of the ice with the inoculation loop. A piece of "ice" will adhere to the loop. Do not distribute the material on the plate. Just place it on the agar and let H. pylori grow on the small drop of defrosted media, undisturbed. Place the agar plates to grow in an incubator with microaerophil conditions (10% CO2, 5% O2 and 75% N2) at 37°C for the weekend.
On Monday, passage the bacteria to a new GC Cholesterol plate
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, and pass them onto a new plate on Tuesday for Wednesday's work, where bacteria are fit enough to be used for infection.
Note
A "plate" corresponds to a Petri dish of 10 cm diameter with ca. 20 cm of agar growth media per plate.
Note
Optional: You can grow H. pylori in a 10% CO2 incubator at 37°C. They will just take a bit longer to grow as they do in microaerophil enviroment.
Seed AGS cells 48 hours before infection will be performed.
Seed AGS cells 48 hours before infection will be performed.
The objective is to have a cellular confluency in the 6-well of 80-90% at the moment of infection. For you to achieve this, you have to know your cells and their multiplication speed. For AGS cells the usual splitting for 48 hours with synchronization (see below) was 1:8 (from a 90% - 100% confluent culture)
Synchronize the AGS cells 16h before infection
Synchronize the AGS cells 16h before infection
Remove serum-containing media (RPMI + 10% Inactivated FCS, Complete Media (CM)) and change it for media without serum (RPMI without serum) around 16 hours before infection, in order to synchronize cells in G0.
Note
For each cell, a different method of synchronization might be needed. For example, I have gotten better results with HEK293 cells by using DMEM + Serum but without glutamine.
Immune cells reacts very strong to lack of serum in their growth media. It can even cause changes in differentiation status.
Resuspend bacteria
Resuspend bacteria
For 1/4 of plate grown bacteria, collect all bacteria using a sterile cotton swab and introduce it in a tube containing at least 500 µl of PBS. Using circular movements, remove the bacteria culture attached to the cotton fibers using the PBS solution. The objective is to have as much bacteria in the solution, and reduce the amount of liquid attached to the cotton swab before discarding it.
Measure the optical density (OD) of the bacterial suspension at 550 nm
Measure the optical density (OD) of the bacterial suspension at 550 nm
Make a 1:100 dilution of bacteria suspension in PBS (10µl bacterial suspension in 990µl PBS) and measure its optical density (OD) in a densitometer at 550 nm.
Calculate the amount of H. pylori colony forming units (CFU) per ml of suspension
Calculate the amount of H. pylori colony forming units (CFU) per ml of suspension
Multiply the dilution factor to obtain the OD550 / ml of suspension.
Calculate the amount of bacteria in your suspension taking into consideration that and OD550 of 0,1 is equivalent to aprox. 3x107 CFU/ml.
Note
The value of CFU/ml in a OD550 of 0,1 tends to vary depending of the wild type strains or mutations. Some variations are negligible. Nonetheless, make sure your CFU/ml is similar to the value given here.
Infect your cells with an Multiplicity of Infection (MOI) of 60.
Infect your cells with an Multiplicity of Infection (MOI) of 60.
Multiplicity of Infection (MOI) of 60 means that you will add 60 bacteria for each cell in the well. For general calculations, each well of a 6-well cell culture plate has approx. 1x106 adherent cells at a confluency of 90% to 100%
Prepare the AGS cells 30 min before infection
Prepare the AGS cells 30 min before infection
Remove the serum-free media from the cells and add 1 ml of complete media (CM) per well around 30 min before infection
Infect the cells
Infect the cells
Infect cells and incubate at 5% CO2 37°C for 3 to 4 hours.
Note
The minimum time I have been able to infect synchronized cells and detect CagA phosphorylation in Western blot is 1 hour at MOI 60.
Stop infection for harvestinfection
Stop infection for harvestinfection
Stop the infection by placing the plate on ice. For the rest of the procedure, maintain the samples cold.
Note
Since H. pylori is a Biosafetly Level 2 bacteria, make sure you can keep the surface of the ice free of contamination before disposing of the ice.
Collect supernatant for cytokine measurements
Collect supernatant for cytokine measurements
Collect the media for cytokine measurements. If no cytokine measurements are necessary, remove the media with help of a vacuum pump. Add 1 ml PBS* (PBS with protease and phosphatase inhibitors). Prevent the well’s surface from drying out (crystal formation damages the cells).
Note
PBS*: PBS with Proteinase Inhibitors (1 mM PMSF, 1µM Leupeptin, 1µM Pepstatin, 1 mM Sodium Vanadat), fresh made
Detach cells
Detach cells
With a cell scrapper detach the cells from the bottom of the plate.
Note
Prevent too much scrapping since this will damage the cells more that desired. It helps to go methodically over the surface of the well. If you look carefully the reflection of the light will show you where cells have been already removed from the surface.
Collect cells
Collect cells
Collect the cells suspension in 1,5 ml tubes. Centrifuge the cells 500 g for 10 minutes at 4°C in a swing rotor centrifuge.
Prepare samples for Western blot
Prepare samples for Western blot
- Discard ALL the supernatant taking care of not loosing the pellet.
- Resuspend the pellet in 20 µl of your favorite Lysis buffer containing protease inhibitors.
- Add immediately 25µl of 2X SDS loading buffer and boil the samples at 95°C for 10 min. To avoid condensation and stickiness of DNA, place the tubes immediately in ice. Do not centrifuge!
Note
If you have problems pipeting the samples on the SDS-Acrylamine gels (or similar) because of clumping of the samples, the placement of the tubes on ice after boiling was not fast enough.
Separation of proteins
Separation of proteins
- For separation of proteins, we use the protocol Single Gel system based in Ahn T et al (2001)
Transfer of proteins to PVDF membrane
Transfer of proteins to PVDF membrane
- Activate gel for detection of proteins using the stainfree system for quantification of proteins present in the sample. These values allow us to do the normalization of the phosphorylation signals after immunodetection.
- For transfer of proteins to the PVDF membrane, we use the protocol Trans-Blot® Turbo™ Transfer with Home-made buffers
Activate PVDF membranes
Activate PVDF membranes
Place the membranes in contact with methanol to activate them.
Note
If you use nitrocellulose membranes, do not use methanol. It will destroy them.
Safety information
Methanol is toxic. Use your Personal Protective Equipment when handling methanol.
Western blot - Block the membranes
Western blot - Block the membranes
Block membrane with TBS-T (TBS + 0,075% Tween 20) with 3%BSA for 1 hour at room temperature.
Western blot - Primary antibody
Western blot - Primary antibody
Add the anti-phosphotyrosine antibody in a dilution 1:10000 dilution (Clone 4G10 antibody from Millipore), for 1 hour in PBS with 3% BSA at room temperature.
Western blot - Remove excess primary antibody
Western blot - Remove excess primary antibody
Wash 4 times, 15 minutes each, with 5 ml TBS-Tween (TBS + 0,075% Tween 20) at room temperature.
Western Blot- Secondary antibody
Western Blot- Secondary antibody
Add 1:10000 anti-mouse peroxidase conjugated antibody (Sigma) for 45 minutes in TBS 0,075% Tween 20 .
Western blot- Removal of excess secondary antibody
Western blot- Removal of excess secondary antibody
Wash 4 times 15 minutes with 5 ml TBS 0,075% Tween 20 at room temperature.
Western bot - Detection
Western bot - Detection
- Remove ALL WASH BUFFER . Take care to not to let the membrane dry in this step!!!
- Add Millipore Immobilon Western (Cat Nr. WBKLS0500) developing solution (1 ml solution A, 1 ml solution B).
- Let the membrane roll with the developing solution for about 1 to 2 min
- Place membrane between to two transparencies.
- Place membrane in Gel documentation machine (Gel Doc™ XR+ Gel Documentation System) and detect the chemoluminiscence
Quantify signals relative to control infection
Quantify signals relative to control infection
Using the software ImageLab from Bio-Rad, set lanes and bands. Quantify signals using relative values to the control infection.
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Normalize values
Normalize values
Using the same image and the image of the Stainfree gel, use the normalization option from the ImageLab software. Multiply the values obtained for the phosphorylation signal with the values obtained for the normalization.