Posts Tagged ‘2D Gel Electrophoresis’

Proteomics Refined

 :: Posted by American Biotechnologist on 05-21-2010

As a proteomics fan in general and a 2D Gel Electrophoresis fan specifically, I have been following the Ludesi Blog which I have found to be a resource rich in 2D information including a knowledgebase, webinars and an great tool that pulls together all proteomics related tweets from twitter. For those of you who are not familiar with Ludesi, they are a Swedish company that was founded in 2001 by Ola Forsstrom-Olsson who was listed in MIT Technology Review’s Top 100 list of the World’s Best Technology Innovators Under 35 in 2004.

In addition to having a great blog, Ludesi produces fantastic 2D gel imaging software and provides an analytical service that is fast, user friendly, compatible with all types of stains, has great technical support and a unique pay as you go system. But the best of all is that the experts at Ludesi analyze the gels for you and save you some serious computer time!

To get started:

– Download REDFIN software at www.expressionproteomics.com/redfin and follow the registration
procedure
– Upload 2-D gel images and purchase credits.
– Each gel is analyzed by multiple professionals at Ludesi.
– Data and gels are stored on the redfin site and can be accessed via the secure server from any computer (anywhere) at any time.
– Since Redfin is a Software As A Service (SAAS) it is automatically updated every time you log in (no need to pay for updates
– Best of all…you only PAY FOR WHAT YOU USE!

For a limited time, Bio-Rad has arranged for new customers to receive three free credits when first trying out the service.

What does that mean? Well, to have a gel analyzed using the basic service (appropriate for almost all researchers) you will need 2 credits and to analyze a gel using the Pro service (removes streaks, and artifacts and expands upon quality assurance for spot detection and matching) you will require 10 credits per gel.

To get your free credits, download redfin from www.expressionproteomics.com/redfin, click on the “use value code” button and enter “TRYREDFIN” and away you go!

To read more about Redfin, see the a href=’http://www.americanbiotechnologist.com/blog/wp-content/uploads/2010/05/Redfin-brochure.pdf’>Redfin brochure

For a thorough review on the limits, benefits, and perspectives of gel-based proteomic approaches see the recently published review article in Proteome Science.

Chevalier, F. (2010). Highlights on the capacities of “Gel-based” proteomics Proteome Science, 8 (1) DOI: 10.1186/1477-5956-8-23

A gel worthy of high-impact publication

 :: Posted by American Biotechnologist on 03-02-2010

Yes…SDS PAGE is boring. But it’s an indispensable part of our bench research. We put tons of time into designing experiments, sample prep and playing video games (oops…Freudian slip). It is therefore absolute torture when we load our precious sample into a gel, run it for 30 to 60 minutes only to discover that the quality of the gel has significantly compromised what promised to be an award winning figure worthy of publication in the likes of Nature or Science (would you even think of publishing anywhere else?).

For as long as I can remember, Bio-Rad has been at the forefront of electrophoresis technology. There is hardly a graduate student out there who has not come across a green MP3 or MP tetra electrophoresis unit during the course of their graduate work. Now Bio-Rad has released a new SDS Polyacrylamide Gel that provides high resolution separation regardless of the origin or complexity of the sample. Moreover, whether you are a 2-D researcher, a “stainer” or a run of the mill 1D separation type of scientist, the new TGX gels will help you produce results that your reviewers will be proud of! See the tech note for more details.

Solution to Ugly Gel Mystery: Week #3

 :: Posted by American Biotechnologist on 02-15-2010

Last week’s ugly gel was one of the ugliest 2D gels that we’ve seen in a while. There was capacious amounts of horizontal streaking across the top of the gel which made it nearly impossible (OK….completely impossible) to discern discrete spots necessary for protein identification.
Some likely causes for such ugliness include:
1. Protein Overloading
2. Proteins not properly and stably solubilized
3. DNA contamination
4. Incomplete focusing

In order to rectify this problem, it is important to ensure that all proteins are completely solubilized by using a strong chaotropic extraction reagent. The concentrations of urea, thiourea, detergents, carrier ampholytes, and reducing agents (DTT or TBP) are also critical. Every sample type typically requires a new sample preparation method. A good starting point for sample preparation includes the following standard buffer solutions:
8—9 M urea, 4% (w/v) CHAPS, 2 mM TBP or 1% (w/v) DTT, 40 mM Tris, 0.2% (w/v) Bio-Lyte (ampholyte), pH 3—10
7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2 mM TBP or 1% (w/v) DTT, 40 mM Tris, 0.2% (w/v) Bio-Lyte (ampholyte), pH 3—10
ReadyPrep sequential extraction kit reagent 3: 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3—10, 40 mM Tris, 0.2% (w/v) Bio-Lyte (ampholyte), pH 3—10
ReadyPrep 2-D rehydration/sample buffer 1: 7 M urea, 2 M thiourea, 1% ASB-14, 40 mM Tris

Allow sufficient time for full denaturation and solubilization. Allow the sample to sit at room temperature in the solubilization solution for 1 hr before applying to IEF.
Remove insoluble protein complexes by centrifugation (>10,000 x g) prior to IEF.

Luckily, Bio-Rad can help you with this as well. Bio-Rad’s ReadyPrep protein extraction kit and sequential extraction kits make it easy to extract the types of proteins you need quickly and cleanly without contamination from DNA or other artifacts. Yes…2D electrophoresis can be complicated, but with the proper techniques you will end up with clean looking gels that provide you a tremendous amount of data for your proteomics studies.

Solve the Mystery of the World’s Ugliest Gel: Week #3

 :: Posted by American Biotechnologist on 02-04-2010

OK Supersleuths. This week’s gel is a toughie. Chances are, you don’t see such a complicated situation every day! Once again, the source is a 2D gel and I am definitley hard pressed to find an answer. As a review, our questions are:

1. Why is this gel so ugly?
2. How did it get this way?
3. How can Adriana prevent this from happening again?

To submit your responses (and receive a free Bio-Rad TGX long life gel in the process)
simply leave a comment by clicking on the comments link below or fill out the ugly gel feedback form. If you choose to fill out the form, you will be eligible to receive a free TGX sample gel compliments of Bio-Rad Laboratories.

gel#3

Help Solve the Mystery of the World’s Ugliest Gel: Week #2

 :: Posted by American Biotechnologist on 01-25-2010

Following fast on the heels of last week’s ugly acrylamide mess, here is a submission from one of our Canadian friends. Adriana from the University of Toronto is hoping that you can help her solve the mystery of this warped wonder. Once again, here are the questions:

1. Why is this gel so ugly?
2. How did it get this way?
3. How can Adriana prevent this from happening again?

To help Adriana, simply leave a comment by clicking on the comments link below or fill out the ugly gel feedback form. If you choose to fill out the form, you will be eligible to receive a free TGX sample gel compliments of Bio-Rad Laboratories.

gel week 2