26 replies to this topic

[ProteaKyle]

When creating a stacking gel I use a 5% stacker with a 200uL overlay of Butanol. Then dump it off and rinse with deionized water. Something that I noticed is that you stated that you used 16uL of sample to 4uL of loading buffer; the general rule of thumb for laemmli buffer is that it must be at least 50% of the final volume. The laemmli buffer is important because of the glycerol in it which increases the density of your sample and allows it to sink in the wells of the gel. Another thing that may be causing the blurred bands is how long the sample sits in the well before you start running it. The longer the sample sits the more it starts to diffuse and less crisp your bands will be. I hope this help you or anyone who may read this with the same problem.

[azrael201]

this is wierd

i too have recently been getting what i think is a leak but after the gel polymerizes there is a thin 1mm space at the bottom.

i have never had a problem making gels and they usually solidify in at most 15 minutes. Yesterday i spent 5 hours remaking gels because they would either leak out of the sandwich or not solidify properly (there are imperfections in the gel which resembled different parts of the gel solidifying at different times).

i am making an 8% gel how long does it usually take to harden?

my APS was only 2 weeks old would it make a huge difference? i also have been ejecting the TEMED down the side of the tube instead of mixing it directly.

i threw out a gel yesterday because i saw the 1mm space at the bottom and after pouring on the stack gel i noticed that the stack gel level fell and i couldn't help thinking it it leaked through my resolving gel. i did noticed new gel formation at the bottom where the space was.

i also mix by turning it upside down and i don't degas...are these potential problems? i mena i've never experienced anything til recently. i started suspecting the brand new bottle of acrylamide:bis solution so i borrowed the neighboring lab's. i think it's working better? i think i've lost all confidence

Edited by azrael201, 17 February 2011 - 12:13 PM.

[mdfenko]

azrael201, on 17 February 2011 - 12:04 PM, said:

this is wierd

i too have recently been getting what i think is a leak but after the gel polymerizes there is a thin 1mm space at the bottom.

i have never had a problem making gels and they usually solidify in at most 15 minutes. Yesterday i spent 5 hours remaking gels because they would either leak out of the sandwich or not solidify properly (there are imperfections in the gel which resembled different parts of the gel solidifying at different times).

i am making an 8% gel how long does it usually take to harden?

my APS was only 2 weeks old would it make a huge difference? i also have been ejecting the TEMED down the side of the tube instead of mixing it directly.

i threw out a gel yesterday because i saw the 1mm space at the bottom and after pouring on the stack gel i noticed that the stack gel level fell and i couldn't help thinking it it leaked through my resolving gel. i did noticed new gel formation at the bottom where the space was.

your problem is probably a combination of old aps and the way you introduce temed to the solution. we usually use freshly prepared aps when preparing gels so that we don't have to worry about its effective strength.

we allow gels to sit for at least an hour before stacking to ensure reasonably complete polymerization. polymerization will actually continue for some time after. when performing protein sequencing from blotted protein, the protocol calls for overnight aging of the gel to ensure complete polymerization (free acrylamide can cause problems with sequencing). if we use weak aps or weak temed (temed turns yellow and loses potency as it ages) then we can see the same effect you describe, but, if we let the gel sit longer then it will complete.

[ProteaKyle]

azrael201

I can probably help you out. I work for Protea Bioscience as a laboratory technician, specializing in gel electrophorsis. I can offer some quick tips but it might be better to know which system you are using? Which brand of Acrylamide:bis solution you use? How much TEMED and APS you use per 10mL of gel solution? However, here are some things you can try. If you are using glass plates, check to make sure there are no cracks or chips in them. I wouldn't necessarily worry about the age of the APS solution. I have used some in the past that were older than 2 weeks and it worked fine. However, it might be a good idea to make more. When making more 10% APS listen to the tube while adding the water to the APS. APS that is still fresh will crackle when water is added. If your TEMED has turned yellow it means it has been oxidized by the moisture in the air and the strength has decreased. The imperfections in the gel are most likely due to the amount of APS and TEMED you are adding and my suggestion would be to try varying those. I would start by decrease your APS and TEMED. If those don’t work respond and we can delve into it deeper.
Good luck,

Kyle
Laboratory Technician at Protea Biosciences
www.proteabio.com

[azrael201]

thanks for the helpful replies. i borrowed the neighboring lab's acrylamide:bis and i think it worked. i'm still not entirely convinced that our newly opened bottle of acrylamide:bis is faulty. Perhaps the first few times it took more than 15 minutes and i didn't realized it but i am pretty sure it was definitely not half an hour to one hour. I doubt waiting that hour would prevent that leak on the bottom.

i used Biorad 33% 29:1. For a 10mL prep I use 6ul of TEMED and 100ul of 10%APS. I cast the gels in a biorad apparatus that clamps the glass plates down on to a sponge for the bottom seal.

I even used my postdoc's reagents including his APS and TEMED and I remember it didn't solidify correctly.

Our TEMED is not yellow yet but maybe it is the APS but it's strange I had no problems making a gel just last week with the same reagents except the new acrylamide:bis.

[ProteaKyle]

If you are sure that everything else is the same then it sounds like it was your Acrylamide:Bis that was the problem. I would double check your Acrylamide:Bis to make sure you have the same ratio of acrylamide:bis that you have been using in the past. There are usually three ratios 37.5:1, 29:1, and 19:1 and the different ratio will definitely affect your polymerizing time. I have read that the optimal polymerization time for overlaid gels is when the visible polymerization takes place in 15-20 min; and the optimal polymerization time for nonoverlaid gels is when visible polymerization takes place in 8-10 min. You can control this by varying the amount of TEMED and APS added. I hope that this helps you out.

Kyle
Laboratory Technician at Protea Biosciences
www.proteabio.com

[azrael201]

can you comment on what happens if there is an incorrect amount of APS and TEMED?

would you observe the differential polymerization or even the leak at the bottom of my gel?

[ProteaKyle]

There are many reasons why the right amount of APS and TEMED are important. The reason why it is important to get visible polymerization in between the times I listed above is because if you don't then oxygen will begin to enter into your acrylamide and inhibit polymerization. You may have noticed this when you think your gel has polymerized and you pull out the comb and the wells have not fully polymerized or did not polymerize at all. Also when you reduce the amount of APS and TEMED it increases the length of the polymer chains, increases elasticity of your gel, and lowers the cloudiness of your gel. Which are all good things. However, if the TEMED and APS are too low then the gel does not polymerize fast enough and the oxygen begins to affect your acrylamide. The most common evidence that there is too much TEMED or APS, is the schlieren pattern. The schlieren pattern is the swirls that can be seen after polymerization. The schlieren pattern can also be caused by the poor mixing of the solution after adding the APS and TEMED. To mix properly swirl the solution approximately ten times. The swirling motion reduces the chance of inducing oxygen into your gel solution. It sounds like you are on the right track for the amount of TEMED you use. The amount of APS you use sounds a little high, but I could be wrong. There are so many things that can affect the how much you use. I think Bio-Rad recommends you start use around 50ul of APS per 10mL of gel solution. Protea on the other hand recommends you to start with 75ul of APS per 10mL. This is because we have a special recipe for our gels that give them a drastic increase in strength over the average gel. So it depends on your recipe but those are some starting places.

The leaking of your gels could be caused by the Bio-Rad apparatus that you are using. Make sure the sponge seal at the bottom is clean. If the gel solution is not cleaned off after every use then the gel solution can gunk up the sponge and you can lose a good seal.

I hope this helps you out. If you want to take a look at Protea's gel solutions and precast gels click these links.

Protea’s Ready to Run Gel Solutions

Protea’s Precast Gels

Good luck,

Kyle
Laboratory Technician at Protea Biosciences
www.proteabio.com

[azrael201]

Hi Kyle,

thanks again for all the help!

i recently attempted the gel again and was much more patient and allowed the gel to sit for 30-60min. I checked it at 30 and it seemed solidified but the excess in the tube was not fully polymerized so I allowed it to sit until 60 minutes.

When I added the stacking gel I noticed that five minutes after filling it to the brim, the walls of the ends would leak down to half the wall height. Is this either too slow polymerization or too much of a leak through the sides of the glass plates?

I checked the bottom of the glass after both running and stacking gels were solidified and noticed there was still a 1mm space at the bottom but interestingly enough the space formed a concave with the apex 1mm up towards the center. I am assuming improvement in the seal?

Regardless of these observations I cannot help thinking it is the acrylamide:bis stock that has loss its original concentration, and thus slowing down the polymerization? Is there anyway to test for this to verify my suspicion?

Thanks

Edited by azrael201, 01 March 2011 - 07:14 AM.

[ProteaKyle]

Hey Azrael201, sorry about the delayed response.

When I have used the Bio-Rad apparatus with the glass plates, I have also noticed frequent drops in the outside well walls. It seems pretty typical and it should not be a big problem unless the sample you are loading is at such a volume that it flows over smaller wall. If it is important you could try a little bit more TEMED to shorten the polymerization time.

As far at the 1mm space at the bottom of the gel, that is probably due to the sponge seal coming up in between the glass plates. I have also seen this before using that Bio-Rad system. This also should not be a big deal. It just means that you can’t run your gel that extra millimeter.

If it is important to get rid of the leaking wells and the 1 mm space at the bottom of the gel then I recommend you looking into precast gels for your apparatus. Bio-Rad makes precast gels for their gel box, however so does Protea and Protea’s cost less. Protea also sells the empty plastic cassettes; these will not leak and do not need a special gel pouring apparatus. I know Precast gels aren't practical for most labs but I throw them out there because they are extremely nice to use and make the gel process much easier. Also when trying to produce a gel for publication, these gels go a long way in producing quality images.

The Bis/Acrylamide stock solutions can go bad, but that is usually after about a year, as long as it is stored at 4°C. It is important that the Bis/Acrylamide solution is stored at 4°C, however once it is added to make the gel solution it becomes a lot more stable at room temperature.

Kyle
Laboratory Technician at Protea Biosciences
www.proteabio.com

[ashsau]

hi all,
I am having a trouble with polymerizing my SDS-Page. I prepared the Acrylamide stock solution and I checked it by adding APS and TEMED into it. And it s polymerizing. But once I prepared resolving and stacking gels they seems not to polymerize. I simply repeated the protocol for thrice and got the same results. APS is fresh and even TEMED. Is there any influence from Temperature for polymerization. Because the current temperature here is around 33 C.

Any possible option is highly appreciated. Because I am having a hard time with this and all my work is jammed right now.

Thanks a lot,
Regards,
ashsau

[mdfenko]

ashsau, on 23 January 2012 - 05:54 PM, said:

hi all,
I am having a trouble with polymerizing my SDS-Page. I prepared the Acrylamide stock solution and I checked it by adding APS and TEMED into it. And it s polymerizing. But once I prepared resolving and stacking gels they seems not to polymerize. I simply repeated the protocol for thrice and got the same results. APS is fresh and even TEMED. Is there any influence from Temperature for polymerization. Because the current temperature here is around 33 C.

Any possible option is highly appreciated. Because I am having a hard time with this and all my work is jammed right now.

Thanks a lot,
Regards,
ashsau

how long are you waiting for the gel to polymerize?

are you mixing vigorously after adding temed and aps? if so then you may be introducing too much oxygen into the solution. oxygen inhibits polymerization (it scavenges free radicals).

are you copolymerizing the resolving and stacking gels? you should polymerize them sequentially.