Try multiplex assay kits and instrumentation from Bio-Rad. GFP is very poorly detected in a 96 well plate. I would use FITC instead of GFP. Your secondary antibody could be conjugated to the different fluorophores.

If you are trying to develop an in-home assay for two proteins, you will have to work at optimizing the concentration of the antibodies and you will have to include standards for both the protein detections. Rather than looking for a fluorimeter with two channels of detection, I would go with a regular fluorimeter which can scan across the fluorescent spectrum. Read the FITC first and then read the cy5 in a separate run. Most common fluorimeters are capable of that and you dont have to look into new instrumentation.

Good luck

1. I think that will not be ELISA (because there is not enzime-conjugate). That will be immunofluorescent assay in microplates.

2. You need use some microplate reader-fluorimetr having min 2 channels or spectral detection. Ask Tecan about Infinite M200-1000. I'm not sure, but may be it is suitable for You.

Try multiplex assay kits and instrumentation from Bio-Rad. GFP is very poorly detected in a 96 well plate. I would use FITC instead of GFP. Your secondary antibody could be conjugated to the different fluorophores.

If you are trying to develop an in-home assay for two proteins, you will have to work at optimizing the concentration of the antibodies and you will have to include standards for both the protein detections. Rather than looking for a fluorimeter with two channels of detection, I would go with a regular fluorimeter which can scan across the fluorescent spectrum. Read the FITC first and then read the cy5 in a separate run. Most common fluorimeters are capable of that and you dont have to look into new instrumentation.

Good luck

Sanath gave you an idea. However, the multiplex kits (Bio-Rad, Millipore, etc) are quite expensive and you need the instrument that may be around 10,000 up. Those kits could be customized and are mainly designed for more than 8-10 and even 23 proteins that you can detect simultaneously.

Sanath's suggestion is also good relating the in home assay and the instrument to read. But most importantly you have to carefully select your primary and secondary antibodies. The primaries should be on different species, such as mouse and rabbit (monoclonal), or mouse and rat, or at least different isotypes. If not, your secondary will recognize both primaries. So, your secondary could be anti mouse, anti rabbit, etc or anti isotype specific (IgG1, IgG2a, etc). You can also have one of the primaries biotinilated and your secondary be Streptavidin-fluorochrome. You can also have a primary PE and a secondary anti-PE.

Check on Jackson Immunoresearch for secondaries. You can also have the option of having directly the primary against your target conjugated to fluorochrome. There are also kits that you can use to conjugate your primary.

In the Multiplex the instrument detects different intensities of same color by a patented system.

I hope this helps.

Thanks all for your response. 

I was thinking to design a in-cell WB assay using a mouse and rabbit antibody. We just need 2 antibodies: protein of interest and the normalization control. We have an odyssey Li cor which read infrared.

Best