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Wednesday, March 26, 2008

Use of polyethylene glycol for drying polyacrylamide gels to avoid cracking

Authors: Amir Moghaddam and Nils Reinton

Furst Medical Laboratory, Søren Bullsv. 25, N-1051 Oslo, Norway (amoghaddam_at_furst.no).

Electrophoretic separation of proteins in non-denaturing and denaturing polyacrylamide gels remains a common technique in life science and discovery laboratories. When polyacrylamide gel electrophoresis is the last step in experimental investigation, the gel is dried down for storage, photography or exposure to X-ray film. There are two common methods for drying down polyacrylamide gels. The first and the older method is to place the gel on filter paper, to cover the gel with a plastic film and to dry the gel under vacuum and heat. Once the gel has dried, the plastic film can be removed. This method has been reproducibly used for decades for gels with low percentage polyacrylamide, such as less than 10%. If analysing relatively low molecular weight molecules, such as peptides, then higher percentage polyacrylamide gels is required and drying these gels becomes irreproducible and sometimes impossible. The gels would often crack. Several recommendations have been made to overcome the cracking problem, mainly by soaking gels in diluted glycerol. High percentage polyacrylamide gels, soaked in glycerol do not reproducibly dry with this method without cracking.

The second and newer method of drying polyacrylamide gels is to sandwich the gel with 2 cellophane sheets and to clamp the whole cellophane sandwich. The polyacrylamide gels needs to be soaked in 1-3% glycerol before drying and the cellophane sheets need to be soaked in 10% glycerol. As the cellophane dries, it shrinks and stops the gels cracking. The cellophane method is much more reliable for drying high percentage polyacrylamide gels than drying on filer paper, although the problem of cracking remains for 12 to 16% gels. However, there is one major draw back. The cellophane cannot be removed from the gel as it is irreversibly stuck to the gel. This becomes a problem if the gel is to be exposed to X-ray film and if the radiolabel is a or b emitter, such as 35S. For example, Metabolically labelled 35S-methionine and 35S-cysteine labelled proteins, separated on a polyacrylamide gel and dried in a cellophane sandwich cannot be exposed to X-ray film as the emitted particles do not cross the cellophane membrane. If fluorography is to be used, then the gels have to be soaked in a fluorophore and that increases the chances of cracking while drying.

We have overcome the problem of drying high percentage polyacrylamide gels without cellophane sticking by simply soaking our polyacrylamide gels in 20% polyethylene glycol (PEG)-400 (1) prior to drying. This technique is reliable and versatile and works with both gel-drying methods, drying on filter paper and drying in a cellophane sandwich. Figures 1 and 2 show the effectiveness of drying a 16.5% SDS-polyacrylamide gel by soaking in a solution containing 20% PEG-400 and 50% methanol for 15 minutes compared to soaking in glycerol.

Fig. 1:

Figure 1.

16.5% SDS-polyacrylamide gel was used for separation of proteins prior to fixing and staining with coomassie brilliant blue. The gels were not destained completely to allow easier photography.After destaining, the gels were soaked in 20% PEG-400 and 50% methanol (A) or 10% glycerol (B) for 15 minutes before placing on a wet 3 mm think filter paper (Whatman) and placed in a gel dryer and dried under vacuum at 75 oC for 2 hours.

Fig. 2:

Figure 2. 16.5% SDS-polyacrylamide gel was used for separation of proteins prior to fixing and staining with Coomassie brilliant blue. The gels were not destained completely to allow easier photography.After de-staining, the gels were soaked in 20% PEG –400 + 50% methanol for 15 minutes (A) or 3% glycerol for 60 minutes before sandwiching between two cellophane sheets. The sheets were soaked in water before hand according to the manufacturer’s instructions (Bio-rad). The gels were dried overnight.


We also tested whether PEG would interfere with fluorography. We found that we could supplement the fluorophore, AmplifyTM (Amersham), with PEG-400 to aid drying of the gel.Radiolabelled proteins on these gels would produce a stronger signal on an X-ray film compared to the same preparation radiolabelled proteins exposed to film without soaking in the fluorophore (figure 3). We could not assess the intensity of bands on an X-ray film, if the gel was exposed to fluorophore alone without PEG, as the gels would consistently crack into many pieces during drying.

Fig. 3:

Figure 3. An antibody and protein-A sepharose was used for immunoprecipitation of its target antigen from cells radiolabelled with 35S-methionine. The immunoprecipitate was boiled in Laemmli buffer and was loaded onto two lanes of a 16.5% polyacrylamide gel, half in each lane. After separation, the gels was fixed. One lane was soaked in Amplify™ supplemented with PEG-400 to 20% and the other in 20% PEG-400 alone. After drying, the plastic sheet was removed and the gels were exposed to X-ray film.

Many modern laboratories are equipped with cellophane cassettes and not heated vacuum gel dryers. For exposure of gels with b-emitting radiolabels to X-ray film, it remains necessary to remove cellophane sheet from one side of gel. In this case, one side of the gel can be covered with a plastic film, that has been cut to the same size as the gel, before putting on the cellophane. After drying the gel, the plastic film and cellophane from one side of the gel can be pealed off to expose the gel (not shown).

In summary, 20% PEG-400 is a good substitute to glycerol for drying polyacrylamide gels. It works with heated vacuum gel dryers and cellophane sheets and allows cellophane sheets to be peeled off if necessary. 20% Methoxy-PEG (poly(ethylene glycol methyl ether)-350 (1) can also be for drying gels with the same apparent effectiveness and versatility (data not shown).

12 comments:

Anonymous said...

I guess most people would ask if the PEG would interfere with down stream mass spectrometry analysis.

Amir Moghaddam said...

The problem with PEG is that it is often not pure. A solution of PEG of a particular molecular weight is in fact going to constitute of a lot of different molecular weights that is going to interfere with mass-spectroscopy. It may be possible to get around this by using mono-disperse PEGs. I.e. very high quality and high purity PEG’s with a unique chain length. Monodisperse PEG really increase reproducibility of assays as well. To my knowledge, there is only one company that provides monodisperse PEGs, Polypure AS (www.polypure.com).

Anonymous said...

The key to drying high % PA gels is to avoid stretching them. Granted, it's been 20 years since I've had to dry a lot of gels for fluorography, but I routinely dried 15% Laemmlu gels, and 12% Bicine gels with nary a crack. If you are drying onto Whatman paper, slide the filter under the gel while it's in the ice water. Takes longer to dry as the filter sucks up water, but as long as the gel is fully dry (check to make sure it's as hot as the rest of the dryer) you're good to go.
Same goes for drying in dialysis membranes. Make sure there's a lot of water in the sandwhich and you're good to go. Gels crack when they are stretched while wet.

Anonymous said...

Do you know if the PEG will interfere with PAG film bonding?
If not, any thoughts?

SciPhu said...

I am sorry, we do not have any experience with PAG-film. We have seen gel shrinkage during drying though and this may cause disadhesion from the film. Please note that his has not been tested, and feel free to leave a comment if you are testing it yourself.

Daniel Favre, Ph.D. said...

Hi,

I am fixing my SDS-PAGE gels in 30% methanol / 10% acid acetic for 30 min. Then I let my gels over night in 5% glycerol in water.

The next morning, I dry my gel at 55 degrees C for several hours, under vacuum : the most important is that there is a cooling system for removing the heat, or that there is a vacuum branched on relatively cold running tap water (vacuum).

The gels do crack if there is no cooling system with the vacum.

Good luck with your SDS-PAGE gels !

Daniel

steppen wolf said...

You're in the Molecular and Cell Biology Carnival -- here: http://skeptalchemist.blogspot.com/2008/11/molecular-and-cell-biology-carnival-4.html

Thank you for your post!

DK said...

None of the gels shown is worth drying. Too ugly/low quality.

Nothing - no PEG, no glycerol - is needed to be added for gel drying without cracking! If you stain with conventional coomassie R-250, take the gel out of destaining solution (something like 7% acetate, 10% MeOH IIRC) and that's it. In 7 years doing this, I haven't had a single cracked gel.

That's for gels between 6 and 12.5%. Above that I imagine glycerol or PEG is beneficial.

Jens jenssenius said...

Interesting that this idea of using PEG for stabilizing the gel during drying is being rediscovered. At least I think it is rediscovery after the paper by Wallevik and Jensenius in 1982 described the idea of drying PEG-treated gels between cellophane using a simple device, which can now be obtained from www.pateof.com - a most exclusive company in the direct service of scientists.
Yours etc
Jens C. Jensenius

Anonymous said...

The solution containing 20% PEG and 50% MeOH works great on even 20% SDS-PAGE. Thanks so much, ScoPhu.

Quentin said...

worked for me as well. 18% sequencing gel, 0.8 mm thick, subsequent drying on cellophane. Agreed on the Mass spec interference, use of mono-disperse PEG strongly advised. used PEG 1500 in my case.

Belle said...

anyone can help me with drying 15mm 15% SDS PAGE gel? I am fine with 0.75mm 15% gel even though i don't add any glycerol or PEG; but when it becomes 15mm, i screwed the gel up.

Thanks a lot