Posts Tagged ‘2DGE’

Improving the statistic significe of your Western Blots

 :: Posted by American Biotechnologist on 09-08-2011

Western Blotting is probably one of the most ubiquitous techniques in the molecular biology lab and relatively easy to perform. Yet many of us have been frustrated with statistically insignificant results or protein bands that appear either too dark or too light to quantitate.

Well, do not despair! There are many things you can do to help improve the quality of your blots and increase your likelihood of obtaining statistically significant results!

In the video below, you will learn about the many factors affecting western blot analysis (such as detection limit and dynamic range limitations of film and overloaded gels) and what can be done to improve your chances of success.

The presentation was given by Bio-Rad Laboratories Field Application Specialist Dr. Sean Taylor as part of an intimate customer training. Some of the references in the presentation may be specific for that particular customer but the general information contained in this presentation is highly valuable to all molecular biology labs.

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.

Ugly Gel Mystery Week #1 Solved!

 :: Posted by American Biotechnologist on 01-21-2010

Last week we showed you a picture of a very ugly gel and asked the following questions:

1. what’s so ugly about this gel
2. how’d it get this way
3. what can be done to prevent this from happening again

Here are your answers:
1. This is a 2D gel so there should be spots throughout the surface of a gel (unlike a 1D gel which has bands). The spots should be well defined and sharp. In this gel the spots are wavy and undefined. The truth is that you can’t really make out much from this gel.

2. In 2DGE, it is very important to have a straight edge on the top of the gel so that the proteins enter the gel in the correct orientation and separate properly in the second phase of electrophoresis. This gel is a hand-cast gel and was not cast properly.

3. In order to solve this problem Overlay the gel with water-saturated butanol (n-butanol, l-butanol, or t-butanol) or t-amyl alcohol immediately after gel casting. These ensure that the gel has a clean, straight top edge. Use the overlay recommended by the manufacturer of the electrophoresis cell.

Or…you could always use Bio-Rad precast gels! This ensures that you are using a perfectly cast gel every time!

Congratulations to all you electrophoresis sleuths who solved this mystery without any outside help.

If you would like your ugly gel featured on the blog where you can tap into the collective minds of our super-intelligent audience for solutions simply email a picture of your gel to avi@americanbiotechnologist.com and we will include your gel in our next blog post.

Stay tuned for our next mysterious gel to be posted soon!

Help Solve the Mystery of the World’s Ugliest Gels

 :: Posted by American Biotechnologist on 01-12-2010

In celebration of the launch of Bio-Rad’s new long shelf life Mini-PROTEAN TGX precast gels we are asking all of our readers to help us solve the mystery of the world’s ugliest gels. Each week we will post a picture of what we consider to be a hideous gel and we’d like your feedback on the following 3 questions:

1. what’s so ugly about this gel?
2. how’d it get this way?
3. what can be done to prevent this from happening again?

You can choose to either answer freely and anonymously in the comments section of the blog or click on this PAGE link (nice use of double entendre) which will take you to a form where you can fill out your answers and contact info to be eligeable to receive a sample of Bio-Rad’s new gels at no cost (open only to US-based customers, Canadian customers should visit the Canadian Biotechnologist which is running the same contest on their site). All answers will be posted throughout the course of the following week. All responses will be accepted (serious, fun, moody…whatever).

Below is a picture of this week’s ugliest gel. Since it is the first week we’ll give you a hint to the type of gel you’re looking at (2D) and to where you might find some answers. Yes…we’re being easy on you this week. Don’t expect such kindness in the future!

gel#1