Differential Analysis of 2D Western Blot Images: Dr. Mike Hart from Summa Health Systems researches ocular pathologies

Posted by in 2DE Knowledge Base, News

To continue in our series of customer profiles, I would like to introduce you today to Dr. Mike Hart who works as a Research Technologist at the Akron City Hospital, Department of Ophthalmology Research, which is part of Summa Health Systems (USA).

Although Dr. Hart’s main focus area is biomarker discovery, him and his colleagues are utilizing a wide variety of molecular techniques to investigate an even wider variety of ocular pathologies. One of these techniques is two-dimensional electrophoresis coupled with Western Blotting for differential proteomic analysis.

Now, before I tell you more about Dr. Hart’s work, I need to just say a few words on the topic of 2D Western Blots and image analysis…

If you have ever attempted to analyze 2D Western Blot images for differential protein expression you will know that the word “challenging” can be an understatement.

As with protein-specific stains, the antibodies reveal only a fraction of the total proteins separated on the 2D gel.

2D Western Blot performed using CF488-labeled goat anti-mouse Ig

If you are interested in a differential analysis, you need to of course match multiple 2D Western Blot images to each other. Due to the often great differences in the 2D pattern, this process can be extremely difficult, if not impossible, for most matching algorithms and requires extensive control by a human operator.

Furthermore, analyzing any 2D gel image with a very small spot count may mean that your default normalization algorithm cannot be applied, so special care needs to be taken in applying a normalization algorithm where all assumptions are being met.

To ensure a reliable analysis of their 2D Western Blots, Dr. Hart and his colleagues send their images to the Ludesi Analysis Center.

Read below for Dr. Hart’s answers to our seven customer profile questions:

How would you describe the overall goal of your research?

We have three goals….Publish, Publish and Publish, but not necessarily in that order. In all seriousness, like any other research lab we have a general goal of furthering the understanding of eye diseases in order to find better ways to treat or prevent such conditions.

What is your goal specifically for your 2D gel based research?

By comparing pre- and post-experimental treatment serum samples (we use serum as our primary antibody), the 2D Westerns assist in identifying potential autoantibodies generated by the treatment. If the animal generates or possesses autoantibodies they will bind to the protein/s found in the lysate that was electrophoresed (sp) in the 2D gel and transferred onto the membrane.

What type of samples are you primarily working with?

Retinal lysate from rhesus macaque and dutch belted rabbit.

What have you achieved by using 2D gels?

Currently we have proteomic results from tandem mass spec that we are in the process of validating by elisa and immunoprecipitation.

What type of Ludesi analysis do you primarily use (REDFIN Solo, Basic, or Pro)?

All of the above!

What made you choose Ludesi’s analysis service?

The results!!! For our purpose (i.e. not quantitation, but present or not present) the results are fast and accurate. We previously used PDQuest from BioRad and imageJ, but we have found that you need to be an expert to run those programs properly. Ludesi provided exactly what we needed at a reasonable price, so the decision to use Ludesi was very easy for us!

How does using the analysis service help you in your work?

See previous answer!

To see all customer profiles click here