June 19, 2020

5 frequently asked questions about protein analytics

By Gregory Hansen, Support Scientist

Our support scientists answer the five most common questions they get about protein analytics. We explain how to study antibody-antigen interaction on the Biacore system, measure fluorescence intensity on the Typhoon imager, and more.

I would like to study an antibody-antigen interaction on my Biacore system. Which sensor surface should I use?

If the antibody is used as a ligand, we would recommend any of our ready-to-use sensor chips:

  • Sensor Chip Protein A: Mostly used for orientation-specific binding of human antibodies through the Fc-region.
  • Sensor Chip Protein G: Used for oriented capture or binding of all human and a wide range of mammalian antibody subclasses.
  • Protein L: Used for site-directed antibody capture. This sensor chip is good for studying a wide range of antibody fragments, including Fabs and domain antibodies.

To choose a suitable sensor chip, consider your antibody species and type. We have three different capture kits you can choose from:

  • Human Fab Capture Kit: This kit includes reagents for capturing human antibody Fab fragments in biomolecular interaction analyses on a Biacore SPR system.
  • Mouse Antibody Capture Kit: Choose this kit if you need reagents for capturing mouse antibodies as ligands in biomolecular interaction analyses.
  • Human Antibody Capture Kit: Use this kit for capturing human or humanized IgG antibodies as ligands in biomolecular interaction analyses.

You could also use a method like amine coupling to attach the antibody to the Sensor Chip CM5 – but unlike the ready-to-use chips and kits we mentioned above, you would need to establish regeneration conditions.

I have a target protein and would like to study its interaction with other molecules. Which sensor surface should I use?

Choosing a sensor surface depends a lot on the protein and the other molecules you want to study. Here is some general advice:

  • If your target protein is tagged, we have capture kits and sensor surfaces for capture via His, GST, and biotin tags.
  • For covalent attachment, we recommend using our Biotin CAPture Kit and biotinylating your protein with a user- and protein-friendly protocol. The Biotin CAPture Kit enables reversible capture of biotinylated ligands, and makes it easy for you to see several molecules bind with your target protein on a fresh unused surface every time. As with all our kits, you do not need to establish regeneration conditions because we provide a protocol and solution regeneration in the kit.
  • Our second recommendation is to use covalent amine coupling with our Sensor Chip CM5. There is one drawback – if you have stable interactions, you will need to establish regeneration conditions so you can reuse the surface.

I use the Typhoon FLA 9500 imager to visualize the fluorescence of 6-FAM tagged DNA on agarose gel. I would also like to visualize and measure the fluorescence of my liquid samples in a 96-well plate. I know the instrument can read and image the titer plate – can it also measure the fluorescence intensity in each well so I can quantify my samples?

You can perform this kind of application using the multistage and the titer plate frame. You need to use titer plates designed for fluorescence applications (i.e., low-fluorescence titer plates). Then you can analyze your data using ImageQuant TL array analysis, which was also created for this type of application. Please note that you cannot pull relative intensities if you perform a calibration curve on the side (i.e., steps of known concentration of your probe).

I would like to transfer my machine and program onto a newer computer (Windows™ 10 Pro). Is this possible? Do we need to install anything on the new computer?

We do have software compatible with Windows 10. Please note that you will need to transfer the flat frame calibration, or you can request a visit from a Cytiva support engineer who can do this for you.

Please contact us with the serial number of your instrument and we will help you transfer your machine.

Where can I find the software installation file for ImageQuant TL?

Type IQTL in the search bar on our website and click the page for the product you are looking for. Click READ MORE in the product description and click software package to download the installation file.

Note: You can only download the latest version of IQTL from our website. To download an earlier version, please contact us.

For questions or more information, you can contact us by filling out the form at the bottom of our scientific support page.