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In protocols for polyacrylamide gel electrophoresis (PAGE) I often see instructions to degas the gel solution by putting it under vacuum for 10-15 minutes before polymerizing the gel.

I usually don't do this, and when I tried it once I couldn't see any difference. So I'm wondering what exactly the degassing is meant to achieve and how big the effect should be.

  • What effect is degassing the gel solution supposed to have?
  • How important is degassing to achieve good gels?

Any literature that examines the effect of degassing would be appreciated.

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2 Answers

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The reason for degassing your gels is to remove oxygen. Oxygen in the gel interferes with polymerisation, slowing it down and making it less consistent, so degassing makes it faster and more uniform.

From the EncorBio SDS-PAGE protocol:

Polymerization is quicker and more uniform if you degas the first three solutions for a few minutes in an Ehrlenmeyer flask on a house vacuum prior to addition of the last three reagents. Molecular oxygen inhibits polymerisation by reacting with the free radical SO4- ions, which is actually the reason why PAGE gels are poured in tubes or between plates and not in open top horizontal apparatuses, as can be done with agarose. Also it's a good idea to layer some isopropanol on top of the gel as this prevents oxygen getting in and inhibiting polymerisation.

Oxygen can also lead to oxidation of protein products, which might be crucial if you then want to extract the products and use them for something else (e.g. Sun & Anderson, 2004).

Finally, having bubbles in your gel can distort the results and make them less reproducible, as the bubbles will not form consistently with each repetition and they disrupt the physical medium of the polyacrylamide. So another purpose of degassing is to ensure repeatability.

The Bio-Rad acrylamide polymerisation info sheet has the best info I could find:

The formation of polyacrylamide gels proceeds via free radical polymerization. The reaction is therefore inhibited by any element or compound that serves as a free radical trap (Chrambach 1985). Oxygen is such an inhibitor. Oxygen, present in the air, dissolved in gel solutions, or adsorbed to the surfaces of plastic, rubber, etc., will inhibit, and in extreme cases prevent, acrylamide polymerization. Proper degassing is critical for reproducibility. Therefore, one of the most important steps in the preparation of polyacrylamide gels is the evacuation, or “degassing” of gel solutions immediately prior to pouring the gel. This is done by placing the flask of gel solution in a vacuum chamber or under a strong aspirator. In some cases, a vacuum pump may be required.

Buffer stock solutions and monomer stock solutions are usually stored at 4°C. Cold solutions have a greater capacity for dissolved oxygen. The process of degassing is faster and more complete if the gel solution is brought to room temperature (23–25°C)‚ before degassing begins. Furthermore, if a cold gel solution is placed under vacuum, the process of evacuation tends to keep the solution cold. Pouring a gel with a cold solution will have a substantial negative effect on the rate of polymerization and on the quality of the resulting gel.

Polymerization in which riboflavin is used as one of the initiators calls for degassing. The conversion of riboflavin from the flavo to the leuco form (the species active in initiation) actually requires a small amount of oxygen (Gordon 1973).

This explains why polymerization initiated primarily by riboflavin can be completely blocked by exhaustive degassing. However, oxygen in excess of that needed to convert riboflavin to the active form will inhibit polymer chain elongation, as it does in reactions initiated only by ammonium persulfate and TEMED. Thus, while degassing is still important for limiting inhibition, it must not be so extensive that it prevents conversion of riboflavin to the active form. For polymerization initiated by riboflavin/TEMED, or riboflavin/TEMED/ammonium persulfate systems, degassing should not exceed 5 min.

A consequence of the interaction of riboflavin with oxygen is that riboflavin seems to act as an oxygen scavenger. This is supported by the observation that the addition of riboflavin (5 µg/ml) to stacking gel solutions containing ammonium persulfate/TEMED initiators results in cleaner, more uniform polymerization at gel surfaces exposed to oxygen (such as combs). The same effect could likely be achieved by more thorough degassing of solutions without riboflavin.

Whether using chemical polymerization (ammonium persulfate/TEMED) or photochemical polymerization (riboflavin/TEMED or riboflavin/TEMED/ammonium persulfate initiators), reproducible gel quality and separation characteristics require careful attention to gel solution temperature before degassing, and to degassing time, temperature, and vacuum. These parameters should be kept constant every time gels are prepared.

Sorry for the long quotes, but they are pasted here in case the original sources disappear.

References:

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I have to say this is a very well laid out answer. In most applications of typical western blots conducted in our lab, we do not degas the gels and the results are very good. We do make the gels in tubes and pour between plates, effectively removing most of the oxygen from the reaction. –  leonardo Mar 22 '12 at 0:36
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I have been running gels with different Acrylamide/Bisacrylamide ratios recently. People usually work with 1:37.5, 1:29 ratios which are commonly used for DNA and Protein gels. I have noticed that when you work with lower ratios 1:200 - 1:500, degassing becomes fundamental to guarantee reproducible resolution of my proteins. If I don't degass the mix in one of these gels some of the proteins I am resolving (which migrate really close to one another) won't separate well enough. Also, the time it takes to polymerize the gel can go from 20 to about 45 minutes if I don't degass the solution beforehand. Degassing solutions with normal 1:29, 1:37.5 crosslinker ratios doesn't, in my experience, seem to have much of an effect other than having quicker polymerization times. Perhaps it makes a difference with lower concentration gels (8-5%), but I honestly wouldn't worry too much about this if I was routinely running 10-15% gels and reproducible resolution wasn't a concern.

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