I was wondering what would be the effect of too high a concentration of APS in an acrylamid gel.
I am also curious to know how people manage their stock of APS (we dilute APS with water at 10% and freeze aliquots of it).
In my experience, too much aps doesn't affect acrylamide gels...after all, if you have too much APS, after a little while it decompose and then it is unable to act. Also I usually store 10%APS in ddH20 in +4..it last around 2-3 months, and maybe at the end of the period, gel polimerizations is a little slower...
This said..if your gel is not for sensitive application, no problem..if it is for sensitive...then..better safe than sorry and remake it. hope this helps
In my experience, too much aps doesn't affect acrylamide gels...after all, if you have too much APS, after a little while it decompose and then it is unable to act. Also I usually store 10%APS in ddH20 in +4..it last around 2-3 months, and maybe at the end of the period, gel polimerizations is a little slower...
This said..if your gel is not for sensitive application, no problem..if it is for sensitive...then..better safe than sorry and remake it. hope this helps
When I was casting my own gels I always made up fresh APS from powder. Too much APS in the gels will have the same effect as adding salt to your gels - they will run slower and hotter.
How high is too high?
APS delivers the free radicals which are needed in the polymerisation. More APS will mostky cause faster polymerisation until you get way over the top. Then residual APS can cause protein oxidization in the gel, affect the chain length in the polymer and so on. If you want to read about the in more details, I recommend reading the technical bulletin 1156 from Biorad, which can be found here (http://www.bio-rad.com/LifeScience/pdf/Bulletin_1156.pdf).
Storing APS for a too long time in the fridge causes decomposition, which I usually made bigger batches (10ml, 10%) and froze it in 500ul aliquots. I thawed the aliquots, stored them in the fridge afterwards and used them within two weeks or discarded them.
As Christian Praetorius suggests APS delivers the free radicals required for polymerisation. Therefore if you add more APS you can speed up the polymerisation process - you can acheive the same by adding more TEMED too. As a standard we always make a fresh batch of 10% APS in H2O - (you can get 100mg tablets and you just add 1ml dH20) - every week. Like Fulvio Celsi states as it goes off your gel polymerisation will slow down, changeing it every week is a little excessive but we've found that to deliver the most consistent polymerisations.
Be carefull with the TEMED. It acts as the catalysator in the reaction and starts the polymerisation of the chains. The more TEMED you add, the more chains you will initiate, leading to a shorter overall chain length. The resulting gel will be much more fragile and harder to handle for staining etc.
10% stock we keep in -20 and take each aliquotes out and maintain that in 4degree.
We make 10% AP in ddH2O and store most of it in about 0.5mL aliquotes at -20. Once we thaw an aliqoute, we keep at 4 degrees for quite a while- (about 2 to 3 wks. I have never tried longer than a month) and it works well.
Yes, I have tried adding a bit more TEMED and/or AP to quicken polymerisation and it has worked well, but like Christian said, how much is 'too much' to cause damage, I am not sure. I increase to quicken polymerisation just because it becomes too slow with low room temperatures in winter.
Interesting comments, thanks.
I ran gels with double amount of APS, and observed abnormal unsharp band at low kDa (~5 kDa).
I repeated the experiment with twice less amount of fresh APS and let you know.
Hi
high ammount of APS may also make your gel to polymerase faster and became harder and less elastic. so the gel became easily breakable and may not polymerise properly. it may also lead to a deformed gel where some places can take more stain than others it may also interfer with protein migration giving a deformed migration pathern. For my experience i used double APS and TEMED amount to faster polymerisation and its showed good results. so my advice to you is that you don't exceed 2X to 3X but for both APS and TEMED. Regarding APS prepartion i used to prepare 10% APS solution that i aliquot on 1 ml solution and freez at -20 °C, the solution was perfectly stable even kept for months and some time i even use the same defrosted solution if i have somthing left from the previous day and its was still fine. Good luck!
thanks for the comments.
What would happen incase one forgets to add APS during preparations?
@Omondi: Nothing, the chemical reaction will not start as the radicals, which the APS provides, are essential for the reaction.
@ Nicolas Luigi Pascal Casadei: So do you think adding more APS caused the lower MW bands to be fuzzy or deformed based on your next experiment? Please update. Thanks
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