REGISTRO DE PROCESOS MUSICALES (OBSERVACION PARTICIPANTE CLASE # 6)

Cells with 70% - 90% confluency in a 75 cm2 bottle were detached using 2 ml trypsin EDTA, and collected in 10 ml RPMI serum-free media. To collect the cells, the suspension was centrifuged at 200g for 10 minutes, washed two times with 10 ml PBS (-Ca2+, -Mg2+), followed by a washing step with 5 ml of FACS buffer freshly prepared. Cells were finally resuspended in 1 ml FACS buffer. Viable cells were counted and 3x105 _{cells added to a well}

from a 96-well plate with round bottom. Cells in the 96-well plates were centrifuged at 210g

for five minutes, their supernatant was removed and 200 µl of a FACS solution with PMSF (1 mM, used as proteinase inhibitor to stabilize the fusion proteins during the incubation) containing 5-10 µg (per 1x106 cells) of the fusion protein to be tested were added per well. Cells were incubated at 4°C for one hour and washed four times with FACS buffer with PMSF, 200 µl per well. Anti-GST primary antibody was added in a dilution of 1:1000 in FACS buffer with PMSF and incubated for 60 minutes at 4°C. To remove unbound antibody, two wash steps were repeated followed by addition of anti-mouse Alexa488 secondary

antibody (1:1000) in FACS buffer with PMSF and incubation in darkness for 45 minutes at room temperature. Cells were washed two times in FACS buffer with PMSF and collected in 500 µl of FACS buffer with PMSF to be analyzed by flow cytometry.

FACS buffer 27 mM Tris-HCl pH 7,4 , 137 mM NaCl, 2,4 mM KCl

2.2.3.18

Production of AGS exudates

AGS cells grown to a 70% - 90% confluency on 12-transwell membranes with 3 µm pores or on 75 cm2 cell culture flask were washed two to three times with PBS (-Ca2+, -Mg2+) and left in 500 µl or 2 ml PBS (-Ca2+, -Mg2+) for 60 minutes at 37°C with 5% CO2. The ideal time

point to collect the exudate was determined by evaluating thechange of the PBS viscousity. The 2 ml exudates from the 75 cm2 flasks were collected and filtrated though a 0,22 µm pore membrane. The filtrated solution was used to induce the expression of the Cag apparatus for EM studies.

2.2.4 Protein work

2.2.4.1

Protein concentration estimation

A BSA 100 mg/ml start solution was used as standard protein concentration. 10 µl of the protein solution and the standards in different concentrations (0,5 – 400 µg/ml), were added to 90 µl PBS and mixed. To this suspension, 1000 µl of Bradford solution was added, mixed and incubated 15 minutes at room temperature in darkness. The protein concentration was measured (OD595) in combination with the standard curve, to determine the concentration of

the samples.

Bradford Solution 0,01 % Coomassie Brilliant Blue G250, 5% Ethanol, 8,5% Phosphoric Acid

2.2.4.2

Separation of proteins and blotting

For the detection of proteins, separation of proteins based on their molecular weight was achieved using a SDS-Acrylamide gel.For immunodetection, the function of certain antibodies depends on the condition of the protein preparations. Therefore, most of the samples were prepared with 2X SDS buffer without β-mercaptoethanol. SDS gels (separation layer) containing concentrations of 6%, 8% and 12% acrylamide were prepared and left overnight at 4°C for full polymerization. Shortly before they were used, the 5% acrylamide upper gel (collection layer) was added. Samples were loaded and gels immersed in SDS running buffer, runned first for 10 minutes with 100V (~ 30 mA per gel), followed by 75-90 minutes at 130V. Proteins were transfered from SDS-acrylamide gels to a PVDF membrane using a semidry electric transfer chamber, with a 1,2 mA/cm2 _{for 75 minutes, using anode I,}

anode II and cathode buffer as transfer buffers. For some gels zinc staining was done using the Zinc Staining kit from BioRad following the manufacturer’s instructions. Once the transfer to the membrane was complete, membranes were dryed and stored at 4°C or -20°C until immunodetection.

SDS running buffer 250 mM Glycin, 0,1% SDS, 25 mM Tris HCl pH 8,3 Anode buffer I 0,3 M Tris pH 10,4; 10% Methanol

Cathode Buffer 25 mM Tris, 40 mM 6-Amino-n-Caproic acid (or glycin), 10% Methanol, final pH 9,4

2.2.4.3

Immunodetection by Western Blot

For the immunodetection, dryed membranes containing the separated proteins were immersed in 100% methanol for no less than 15 seconds. Once wet, the membranes were blocked with 5 ml blocking buffer for one hour. The buffer was then exchanged for 5 ml reaction buffer containing the primary antibody. Incubation of the antibody followed for one to two hours at room temperature. The excess and unbound antibody was washed five times with wash buffer every 10 minutes. 5 ml of reaction buffer containing conjugated secondary antibodies (alkaline phosphatase or peroxidase conjugated) were added to the membrane for 45 minutes at room temperature. Washing steps were repeated three times followed by detection of the secondary antibody. In many cases, re-evaluation of results by detection of other proteins was necessary. For this purpose, PVDF membranes were exposed to 5 ml of 50 mM NaOH for 1-2 hours in order to remove the antibodies. After the stripping of the membrane with the NaOH solution, the rest of NaOH was removed with distilled water and the immunoblot was started again with 5 ml of blocking buffer. A maximum of four stripping processes could be done without observing any damage on the proteins or membrane.

Blocking buffer PBS, 5% Fat-free milk (powder).

Reaction buffer 0,9% NaCl, 10 mM Tris HCl pH 7,4; 0,75% Tween 20, 1% fat- free milk (powder)

Wash buffer 0,9% NaCl, 10 mM Tris HCl pH 7,4; 0,75% Tween 20

2.2.4.4

Fluorescent staining of proteins

Proteins which were to be stained using an AlexaFluor compound, were dialyzed previously in 2 liters PBS (+Ca+2, +Mg+2) at 4°C minimum for two hours in order to eliminate any traces of Tris contained in the storage buffer. After dialysis, staining was made following the manufacture’s protocol (Invitrogen / Molecular probes). Conjugated proteins were store at - 20°C until their use.