Charles, have you got your answer yet? If not, here is my input from my own experience. I do not want to comment on the APS/TEMED/Acryl/buffer/SDS concentrations/pH or the temperature of operation; I expect these basic stuff to be normal. It simply has to be something that your unsuspecting eyes have not caught yet. Going by my experience, I guess you are using a BioRad gel-casting system which requires a grey-colored pad on which you stand the plates (within the green frame) upright. If so, did you try to see what happens after the gel has polymerized (and shrunk)? You would see a build-up of semi-polymerized gel at the bottom and behind the plates. If so, examine the grey pad on which you sit your plates. Do they look rough? Even if they look smooth, stretch them a bit and try to see any roughness. If you do, throw them and simply buy a fresh pair of pads. My problem was this, and I half-solved it by doing so. The remaining half of the problem was solved when I bought fresh lots of APS and TEMED (my APS bottle had a hole in the top corner and was patched-up with tapes). APS should be kept air-tight, and always make fresh solutions. If you are a bit lazy like I am, make about 10ml of APS 10% in water, make 200ul aliquots in simple PCR tubes, dump inside a 50ml conical, and throw in your top freezer. Take an aliquot each time, and you will never have this problem again.

Maybe when you add water on the top you do it too quickly or anyway too strongly so that mix with the gel solution in the upper part, so only the lower part is concentrate enough to polimerize

But if the gel is really 1 cm in height....the problem is that it leaks

This has failed on me multiple times and I am positive it is not a leak. I wouldn't rule out that I am adding the water too quickly. I will attempt to add the water a little slower and more gently. Thank you Annunziata. What does any one else think?

We add water saturated butanol (butanol top layer water below, pipette from top layer) to the top of the separating gel right after we pour it and there is never any shrinkage when we dab the butanol away after the gel is cast and right before adding the stacking gel. We over load the stacking gel so all the lanes in the comb are fully formed correctly. We never have any shrinkage in our 10%-12% sds gels doing this procedure, unless the whole rig leaks, which is visually obvious at the bottom of the gel.

I add isopropanol to the gels to smooth them, isopropanol will not mix with the gel solution

It possible that the gel had leaked from the back of the gel plate. While it is normal that the final volume of the gel to shrink a little after polymerization, the amount of shrinkage you've described is more indicative of a leakage, so do check again.

On another note, I usually add alcohol (preferably water saturated) of about 1ml volume to level the gel.

To my mind..its a problem of polymerization or protocol error.Once the reaction mixture is ready, add a TEMED, following by casting with in 20 seconds,Add water as much needed to make sure the smoother out surface.

Check out the APS expiration dates.

Try a bench experiment -that is mix all contents, leave it for 1 minute in the tube ..and check the solidification strength,and finally the gel caster must be dry ..

Good luck

use less amount of TEMED and persulfate, cool your apparatus before gel pouring

Take every time fresh APS,and add less TEMED for ur experment.

and please let me know details about the buffers you used.

Fill more solution: due to the high quantitative of acrilamide that you use (respect to a 8% i.e.), is a constant procedure to make more "resolving gel" because of lost in volume (due to high concentration). So fill the solution a little bit (1 cm) more of normal one. I used water to cover the gel during polimerization, but I have change with methanol and I have no problems. If the problem is a shrink of gel cast (but I have understand that this is not the case), put a little bit of Temed on the basis of gel caster.

Yust a curiosity: what is the ratio of APS/Temed that you use?

Below may useful for troubleshooting.

Http://aldrin.tripod.com/index-3.html

For 5mls of resolving gel, I added 50ul of 10% APS and 4ul of TEMED. I leave a sample of the gel in a tube and wait for that to solidify to know that the gel I cast has solidified. The buffers are standard pH8.8 and pH6.8 1.5M Tris used respectively for bottom and top gels. The same exact buffers are used by my co-workers with no gel casting problems, so the buffers shouldn't be the issue. I am going to attempt to fill the gel to a higher volume tomorrow and I will attempt using the isopropanol. I will post tomorrow to let you know if it worked. Thank you for all of your responses.

In In how many time your gel get polymerized? If it is in less than ten minutes is due to a TEMED excess and this usually means that the gel didn´t get the time to polymerized properly and that leads to a release of water from the gel (like a dehydratation).

This sounds like a leaky setup to me. Water is a strange choice to top off the gel. I would think it would just dilute all your ingredients. I pour the solution and immediately add isopropanol over the top and have never had this issue. This will help if your issue is evaporation or dilution but not if your setup is leaking. Fill with isopropanol right to the top come back in 5-10 mins, if the isoprop is lower, you have a leak. If you have a leak, figure out what you do differently to your lab mates, otherwise add a strip of para film below your glass plates when you setup, that should help.

check your pH of your upper and lower buffer and add TEMED to a particular volume rather than adding randomly. Allow lower one to set well and when u add butanol you have to rinse it with Distilled water properly and then add upper buffer and allow it to solidify for longer time.

I think it has to do something with your gel polymerisation time. Your gel taking longer casting time could be a reason. Weigh and use fresh APS everytime and check the quality of the TEMED (if it has turned yellow it is of no use). Layering with water will not affect your gel cast or push it down. Monitor your gel casting by leaving a small aliquot of your gel mixture in a tube or beaker. If your gel casting time is too long, revisit the quality of your stocks.

In my view shrinkage is because of DEHYDRATION.

let the gel soak in buffer for a while before loading samples. Otherwise the samples may leak into neighboring wells.

If the buffer contains high percentage of ethanol or methanol, it would cause the problem.

How much water do you add above the gel? If this is a lot (and if you don't put it on very careful) you are going to mix it with your acrylamide solution (which is also mostly water + reagents). Then the local concentration of acrylamide/APS/TEMED can be so low, that there will be no polymerization in the top 5-10mm of the gel. When you then remove your "water" you also remove the non-polmyerized gel and then you loose heigth.

Better use something more unpolar like isopropanol or butanol (although the last one is a bit smelly). This will not mix as good with your reaction solution as water and fulfills the purpose of shielding the reaction from oxygen (which also prevents polymerization) pretty nice. For a ini gel I use some around 150-200ul of isopropanol.

I use water after adding resolving gel with no problems whatsoever..no shrinkage issues with me...i suggest it might be a leaky set up..so use parafilm to seal off the bottom of the glass plates...to a 10 ml 10% resolving gel u should add 4microL TEMED, not more, not less...it should take around 8-12 mins to polymerize.. i usually add around 75% of the length of the glass plates(shorter one) so that even if it shrinks i still have enough R.gel to run my samples!

Good luck mate!

If the gel polymerisation is too fast, it could be the reason. Just like Silvia explained.

I have seen some shrinkage in the past with high percentage gels, and I just attributed it to contraction of the gel matrix following polymerisation, even with a buffer saturated butanol overlay (I use a 5:1 mix of 1x running gel buffer/butanol mix) rather than dehydration. However I have never seen the level drop by 1cm!

I might suggest that what may be happening is that it is the water overlay somehow affecting the gel so that either

1 The water is being added to quickly and so mixes with the top of the gel so it is not polymerising in the upper regions of the assembly or

2 The gel is somehow forming a kind of gradient, and is sinking leaving a larger volume of the aqueous layer.

how much water do you put on the gel, and does the total volume drop (gel + aqueous overlay)?

The shrinkage you're experiencing is very likely due to dehydration. The same has happened to me with DGGE gels. This will happen to greater or lesser extents depending on some of the factors that others have mentioned, i.e. APS and TEMED concs. The temperature of the room in which the gel is setting does have an effect, so you'll find that in hot weather the gel will set quicker than in colder temps, even when all other conditions are the same. I wouldn't use water on the top to smoothen out, this can add to your problem. Instead try adding butanol. This will give a nice smooth line and will not mix with your acrylamide or draw water from it. Afterwards, when the gel is set, tip it out and rinse the area with a little water. Voila.

Charles,

It sounds like the number of possibilities presented so far could all be the cause of the gel shrinking. We have worked with PAGE for a number of years. Generally we always would see some shrinkage over time especially with high percentage gels. It is likely the result of the polymerization process and dehydration. This could be particularly true if the gel is polymerizing too fast. As Rathish stated earlier, you could check the polymerization time of the gel in a separate tube. Fast polymerization also creates heat that could add to the dehydration.

However, from your description, it seems that the shrinkage is localized to where you add the water. If you are not seeing similar shrinkage along the edges or bottom, it is probably due to the addition of water diluting the PAGE. If the PAGE becomes too dilute, then it may not fully polymerize along the edge where you see the 1 cm loss.

I will be out of town next week. So it will be a few more days before I can get to my western. Thank you for the suggestions and I will post a response once I attempt this again.

It is very normal that up to the level you've put the acrylamide mix, after polymerization it will shrink a bit. So put a little higher level whenever you cast it. Amount of Water is not creating any problem here. just make the acrylamide mix littile amount more and pour it. I think it'll solve your problem.

Hi Charles, Hope you are back in your lab and tried western. I will share my experience with you... Well, in accordance with another fellow Christian Praetorius, I am too using - Isopropanol (IPA) on top of the seperating gel (off course little qty like 800-1000uL spread over the full top layer of the Sep gel. It will resolve the problem. Also check if you leave your Sep gel for longer time (few hours) or if you leave it in open or dry atmosphere, if it is so then, avoid it your problem will be reduced itself...... Well, otherwise try IPA. Good Luck.

A little shrinkage in resolving/separating gel is normal, we often don't care for that when are doing one dimensional PAGE, because it is then covered up and sealed by stacking gel. This shrinkage is generally due to the immiscible water (probably due to impurities) in the mixture which is removed with overlayed water and results in the little decraesed volume. Use milli-Q water for the gel preparation and slightly overlay the water (milli-Q, off course) avoid disturbing the gel solution.

Shrinkage in stacking gel is troublesome but it can easily be avoided by transferring the gel into the tank and covering it up with buffer as soon as the gel polymerizes (15-20 mins, normally).

Dehydration of acrylamide gel is common. But leave a wet towel on the surface of gel so that it avoid folding/ thinning of gel

Purnachandra Nagaraju Ganji: There is no reason, why ready-made buffers should be any better - if you know what you are doing. the pH will influence the way the gel runs and also the resolution, but I don't see a reason that it will influence the shrinking.

In my opinion, shrinkage of gel to a little extent is quite common but if it is to a larger extent like 1cm or so then it should be taken care of as the total area of separation would be shortened. There are several factors which may cause such issues.

1. The temperature of the room should be optimal (25-27 degree Celcius). Sometimes you need to put some extra light source like lamp to boost up the polymerization process.

2. You need to check the concentration and % of TEMED and ammonium persulphate used for polymerization.

3. The water you use as the overlay of the separating gel (to avoid air contact and oxidation), you should use water saturated butanol. This avoids mixing of trace water with the gel and further its evaporation leading into shrinkage.

By taking these factors into consideration, one can avoid shrinkage to a great extent.

Charles, have you got your answer yet? If not, here is my input from my own experience. I do not want to comment on the APS/TEMED/Acryl/buffer/SDS concentrations/pH or the temperature of operation; I expect these basic stuff to be normal. It simply has to be something that your unsuspecting eyes have not caught yet. Going by my experience, I guess you are using a BioRad gel-casting system which requires a grey-colored pad on which you stand the plates (within the green frame) upright. If so, did you try to see what happens after the gel has polymerized (and shrunk)? You would see a build-up of semi-polymerized gel at the bottom and behind the plates. If so, examine the grey pad on which you sit your plates. Do they look rough? Even if they look smooth, stretch them a bit and try to see any roughness. If you do, throw them and simply buy a fresh pair of pads. My problem was this, and I half-solved it by doing so. The remaining half of the problem was solved when I bought fresh lots of APS and TEMED (my APS bottle had a hole in the top corner and was patched-up with tapes). APS should be kept air-tight, and always make fresh solutions. If you are a bit lazy like I am, make about 10ml of APS 10% in water, make 200ul aliquots in simple PCR tubes, dump inside a 50ml conical, and throw in your top freezer. Take an aliquot each time, and you will never have this problem again.

I agree with Anil and everyone here as it's a common occurrence. Also, if it's a leakage problem you can add a polymerized stopper at the bottom of the glass cast, which is just made up of your separating gel with a higher percentage of APS.

An easy and cost effective solution for me was to take two pieces of Kim Wipes, fold them to the width of the rubber strips on the cast system and placing them UNDER the rubber. This produces more force against the "clips" as these springs weaken over time do to use.

This has been great for me assuming all other "issues" are mitigated (i.e. chipped glass, etc...).

Hi

Try to keep your pH of upper and lower gel buffer perfectly. Add TEMED a little less rather than your usual load. Then everythnig will be ok. Try to conduct your preparation in a air-conditioned room. best of luck.

For the gel overlay I use 5% SDS in water and do not see significant shrinkage/loss of gel. Plain water will dilute out the top of the still liquid gel and that could lead to a loss in polymerized gel.

I agree with Anil. I also use Bio-rad gel casting system and also had similar problems as Anil mentioned. I noticed that the amount of gel that leaked couldn't be seen from the front end until you remove the whole casting apparatus from the frame. I think in your case may be its just the leaking that you didn't notice properly. In addition to check the gel pads, check the edges of the glass plates if they are even to stop the leaking. Also check if your gel solidifies properly, i.e., within 5-10 mins after adding TEMED. Instead of pouring them for casting, leave them in your mixing flask to see if it solidifies completely. If your gel does not polymerize properly within that time frame and you see some liquid is still there when you slightly tilt the flask, there is definitely some problem with your reagents. You probably have to make fresh reagents including APS and TEMED. By the way, make sure you store your bis-acrylamide solution, APS and TEMED in dark bottle to avoid photodegradation. DO NOT store your TEMED at 4C, instead store it at room temperature. Another unlikely problem could be the bis-acrylamide concentration in your casted gel. Probably the final concentration of the gel is much too less than the desired 10-12%. I am sure you have checked your calculations several times, but just for the sake of being thorough at every steps, if I were in your situation I would definitely check my calculations with someone else in the lab. Even a great scientist can do silly mistakes in calculations. Also, instead of water, use isopropanol to smoothen the top of resolving gel and let it stand till the gel polymerizes. Pour off the isopropanol completely once the gel gets solid prior to adding stacking gel.

I agree with Rajendran Chellaiah.

It also happened to me several times. Check pH of the reagents, use a proper water, always make fresh APS.

I think the main reason of gel shrinkage is too fast polimerisation. Try to make less concentrated acrylamide and to add a lower amount of TEMED and APS. Also, do not mix the final gel solution too much or too long. It is enough to mix just few times up and down very gently, prior to pouring the gel. Do not shake the aparatus aside to check gel polimerisation. Too fast polimerisation results in a higher temperature generation. And when the gel cool down, the polymerized acrylamide reticle shrink. It cause that some amount of water drain out of gel.

People are often afraid if the gel will polymerize and make too concentrated reagents and speed up polymerisation.

It is like pizza. Do it with less care (and stuff) and it will be better. It is a joke, ofcourse.

Best regards.

Direct exposure of Resolving gel results in the drying of the gel resulting in reduction of the size or shrinking whatever it is termed. Hence, covering with organic solvent such as isopropanol or n-butanol after pouring the resolving gel will take care of the shrinkage issue. Hope this helps

If the biorad assembly is dry when you add the gel, then a small amount of liquid is drawn between the plates by capillary action and it appears that the gel has shrunk.  Fill your assembled plates with deionised water for 10 minutes (it also checks for leaks!) and then drain them and put some tissue in between them to dry them then add the gel and it should polymerise well.  Also a lot of people have said to overlay with water saturated butanol, which is also better as it controls the polymerisation.  I am a bit obsessive so I used 1x resolving gel buffer saturated butanol.  

good luck

Just optimise your addition of TEMED

Its given clearly in Molecular Cloning Manual the amount of each component to be added for different concentrations of SDS PAGE Gel. What do you mean by optimising the addition of TEMED? He can check if the TEMED he is using old one or replacing with a brand new TEMED for formation of gel. 

I was experiencing the same problem and found that degassing solves it. I mix all ingredients except APS and apply vacuum for 5-10 min while stirring; then add APS and our immediately.