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Archive for the ‘ugly gels’ Category

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

Solution to Ugly Gel Mystery Week #2

 :: Posted by American Biotechnologist on 02-01-2010

Last week, at the request of our friend Adriana from the University of Toronto, we posted an ugly gel mystery which she needed help solving. Thanks to our educated readership, we were blessed with a multitude of answers. Behold the power of social networks!

The answers included comments from….

Nicole Harris who said “It looks to me like the gel did not set smoothly. Possiable knocked while setting or started to set prior to pouring, causing ripples to form.”

Kirk who said “There maybe within the gel a electro magnetic polarization of molecules responding to natural magnetic fields that developed while curing. A molecular analysis studying the differentiation between the dark and light areas.”

And Dan Moran “Looks like a protein overload first. Then it looks like it was terribly over heated due to the use of the wrong buffer in the wrong gel resulting in high resistance, boiling buffer and melting gel.”

Now here’s an answer from our friendly technical support reps, David Palmer and Cecilia Carino of Bio-Rad Laboratories who gave Andrea the following detailed response:

1)Some sequencing users (yes…this is a sequencing gel folks! ed insertion) apply an excessive amount of power during the pre run to achieve rapid warming of the gel. More heat is generated than can be efficiently distributed by the system under these circumstances. A better (and faster) way to warm up the gel would be to preheat the buffer in a microwave before adding to the IPC. Half the buffer can be heated to almost boiling and then added to the other half at room temperature resulting in a temperature very near the desired 50-55 degrees. Samples can be loaded immediately in this protocol.

2) If the gel reagents are old, have been incompletely mixed, or were not degassed prior to pouring there could be incomplete polymerization along the sides. This in turn could affect the flow of electrical current such that an inordinate amount of heating occurs in close proximity to the spacers. It might be that the first gels you run were cast with fresh reagents, and over time, the reagents have became old. Check and make new reagents if needed.

So….check your reagents people. Old reagents that have been sitting on the shelf for months (you know who you are) should be replaced. Making buffers is a pain, but it’s a much bigger pain to run you precious samples in a gel only to find out that everything has been lost!

As you can see, there’s a real power in “turning to the masses.” Keep sending in your ugly gels and we will do whatever we can to help you solve your ugly mystery!

Stay tuned for another ugly gel to be posted later this week.

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

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!