Detection Methods

If the expressed proteins or phenotypes of interest can be detected with fluorescent reagents, transfected cell microarrays are viewed and photographed with a fluorescent microscope or a laser scanner. If detection is based on radioactivity, slides are fixed and the signal detected with film or emulsion autoradiography. A "Western blot" protocol was developed to detect phenotypes or expressed proteins after transfected cell microarrays are carefully transferred to a nitrocellulose membrane.

Immunofluorescence

All steps are performed at room temperature. Blocking steps and incubation with antibodies take place in a humidified chamber.

1) Fix cells.

2) Permeabilized cells by placing slide for 15 minutes in PBS containing 0.1% Triton X-100.

3) After permeabilization, rinse each slide twice with PBS.

4) Block slide for 1 hour with 500 µl of blocking solution (1.0% BSA/1:100 normal donkey serum in PBS).

5) Remove blocking solution by vacuum aspiration and add 500 µl of primary antibody diluted in antibody dilution solution (0.5% BSA in PBS) for 1 hour. Do not rinse after removing blocking solution. If using two primary antibodies, add them together. Perform incubation in a humid chamber.

6) Wash slide 2 X 5 minutes in PBS.

7) Re-blocked slide for 20 minutes with 500 µl of blocking solution.

8) Remove blocking solution and add 500 µl of secondary antibody in antibody dilution solution for 40 minutes.

9) Wash slide 3 X 5 minutes in PBS.

10) Mount coverslip using vectashield (Vector Laboratories) and store in the dark at 4ºC.

Notes on imaging: Microarrays can be imaged with a conventional fluorescent microscope or a laser fluorescence scanner. We have used a ScanArray 5000 (GSI Lumonics) that can image with a resolution of up to 5 µm. GFP and Cy3 emissions are measured separately after sequential excitation with light wavelenghts normally used for FITC and Cy3 fluorophores, respectively. Slides CANNOT be mounted when using this particular scanner as they will not fit in the entry port.

Below is a laser scan image (10 µm resolution) of a large array expressing GFP, HA-GST and V5-PDGF receptor.

Click here for a high resolution picture and further details

Autoradiography

We add radiolabelled compounds directly to the cell medium containing the microarrays. We added the compounds we tested for 1 hour before rinsing the slides once with room temperature PBS and fixation. Before autoradiography, slides can be processed for immunofluorescence and imaged with a laser fluorescence scanner or a fluorescence microscope. We exposed slides to tritium sensitive film (Hyperfilm cat# RPN 535, Amersham) and developed it as recommended by the manufacturer. When used, autoradiographic emulsion (EM-1 cat# RPN 41, Amersham) was also processed as described by the manufacturer.

Shown below is an example of autoradiograhic detection. Plasmid DNAs were printed in the following order: columns 1 and 5 = pBABE CMV GFP; columns 2 and 6 = dopamine D1-receptor upstream of IRES-GFP in pMSCV; columns 3 and 7 = 5HT1A-receptor upstream of IRES-GFP in pMSCV; and columns 4 and 8 = 5HT3A-receptor upstream of IRES-GFP in pMSCV. GFP expression was detected by laser scanning using a FITC filter. Image in panel 1 below is colored by intensity of signal. Note the much higher level of GFP signal obtained when expression is driven directly from the CMV promoter compared to from an IRES element.

Panel 4 shows an emulsion autoradiograph that detects binding of the dopamine D1 antagonist 3H-SCH23390 to the D1 receptor. Panel 5 shows an equivalent array in which cold SCH23390 was added to the cell medium 1hour before adding 3H-SCH23390. Panels 2 and 3 are high magnification images of the top left subgrid from panels 4 and 5, respectively. The grid in panel 1 is the same as shown in panel 2. The subgrids in panels 4 and 5 are bracketed by penmarks that are fainty visible.

Transfer to nitrocellulose and "Western Blotting" of transfected cell microarrays

We have developed an alternative to immunofluorescence for detecting protein expression in the transfected cell microarray. We transfer the arrays onto nitrocellulose membranes (0.45µm pure nitrocellulose membrane; cat. 162-0116, BioRad) and detect the proteins with standard western blotting protocols. The figure below is an example of an array of myc-tagged proteins detected via enhanced chemiluminescence using a standard anti-myc western blotting protocol. The middle two rows (horizontally) are printed with half the amount of the expression construct as the top and bottom rows. The signal was detected with Kodak Biomax MR film and each spot is ~150 um in diameter. Other methods compatible with proteins affixed to membranes (e.g. far western and southwestern blotting) may also be work with our system, but we have not test this possibility.

Click here for higher resolution version of this image

Transfer procedure:

1) When the cells are ready to be processed, use forceps to lift the slide from the culture dish and quickly rinse it with PBS in a Coplin Jar.

2) After the rinse, remove excess PBS from the slide by briefly blotting its edge with an absorbent paper towel.

3) Place the slide with the cells facing up on a flat surface, immobilize it with tape and allow it to dry for 2-3 minutes (this time can vary depending on how much PBS remains on the slide).

4) Very carefully place a nitrocellulose membrane (about two to three times the area of the slide) on the slide, in a similar manner as is done for traditional plaque lifts (i.e. center first). It is very important to not permit any horizontal movement of the membrane or slide at this step.

5) Keep the membrane on the slide for 1-3 minutes or until the PBS has wetted the entire area of the membrane that covers the slide. Do not press down on or roll a pin over the membrane as this will invariably cause the membrane to slip and destroy the array. Also, do not allow all the moisture on the slide to be transferred to the membrane as this will cause the membrane to stick to the slide and it will tear when it is lifted off.

6) After transfer, carefully lift off the membrane from the slide surface with forceps and allow it to air dry for 2 hours.

7) After drying dip the membrane into a pH 11 CAPS-methanol transfer buffer for 1-2 minutes and transfer it into a standard western blot blocking solution. CAPS-methanol transfer buffer: 2.2g/l CAPS, 10% methanol, pH 11 (usually requires 3-4 NaOH pellets/liter for this pH).

8) Process the membrane with primary and secondary antibodies as in any standard western blotting protocol.

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*Table of Contents *Sabatini Lab *Whitehead Institute for Biomedical Research*