A handy calculator for making SDS-PAGE gels if you are still making your own:

http://www.changbioscience.com/calculator/sdspc.htm

Simply input the total volume, it will do all the calculations for you.

hi guys

could anyone help with a slightly unrelated problem: i get dreadful streaming on the gel (and western, therefore) with SDS lysates, which are absent on CHAPS and MPER lysates, all of breast cancer cell lines. would be grateful to know what protocol seems to give good clean lysate with SDS lysis buffer (re, specific steps such as sonication or heating etc).

cheers!
sean.

I think the protein concentration of your sample may be too high. Or is your sample in high salt concentration which can make the gel ugly?

Doan Lan on Jun 3 2002, 05:04 AM said:

Blur y bandas de distorsión puede provenir de una gran cantidad de sus proteínas. Por lo general, yo uso el 5% de apilamiento y el 13% de gel de separación sin ningún problema. Después de añadir APS y TEMED, se debe verter rápido, a fin de obtener el gel y polimerizadas



Go Debt Ltd

For the distorted bands the reason may be:the amount concentrations of the components may be incorrect according to the protein;may be the running buffer cannot be able to run the sample;may be the voltage is improper and the pore size of the gel is small
so check out for these objectives.
And after making resolving gel,we pour water to know whether gel is settled or not so if there is any thin layer of water in the end then do soak it with filter paper because we dont want water in the gel.
And to settle the gel properly mix the APS and TEMED in the end and very fastly,it enhances the settling of the gel in less time.

For further queries:
<a href=
http://www.wiziq.com/tests/biology>biology test papers</a>


Regards
Rupam Mittal

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.

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

azrael201 on Thu Feb 17 20:04:57 2011 said:

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.

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

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.