does inccreased amount of TEMED and APS affect the resolution of the polyacrylam - (Jan/06/2009 )
does inccreased amount of TEMED and APS affect the resolution of the polyacrylamide gel or mobilityof samples?
if you always allow the gel to fully polymerize then no, they should not affect mobility or resolution.
but, if you tend to use the gel when it first appears to be polymerized then yes, because increasing temed and aps will cause the gel to polymerize faster and will be further along polymerized.
I am wondering too. Less amount or less polymerisation is understandable, but what if you add a little too much of it? Which I am inclined to do everytime I have to cast the gel, what with coffee time always looming
adding more will cause polymerization to proceed faster (and hotter). the gel will reach a stable condition more rapidly and the samples should run well.
how much more are you talking about?
how much more are you talking about?
Not much much.. Rather than 12 ul TEMED, something like 25 ul, and 10%APS also double to what is required in my procotol (acc. to mixture volume). I do it without apparent problems. But I wonder if there is something wrong with it, or if I can add a bit more during those times when gels start delaying in polymerisation for no identifiable reasons
The best way to tell for your specific situation is to do a bit of empirical science. Pour two gels, one with normal amounts and the other with double doses, load identical samples on both, and see what you get.
how much more are you talking about?
Not much much.. Rather than 12 ul TEMED, something like 25 ul, and 10%APS also double to what is required in my procotol (acc. to mixture volume). I do it without apparent problems. But I wonder if there is something wrong with it, or if I can add a bit more during those times when gels start delaying in polymerisation for no identifiable reasons
you could but mixing it between the plates will be difficult. i would just let it sit until it polymerizes or remake the gel (and solutions).
The polymerisation reaction is a vinyl addition catalysed by free radicals. The reaction is initiated by TEMED, which induces and accelerates free radical formation from ammonium persulphate (APS). The free radicals transfer electrons to the acrylamide/bisacrylamide monomers, radicalizing them and causing them to react with each other to form the polyacrylamide chain.
In the absence of bis-acrylamide, the acrylamide would polymerise into long strands (forming Linear Polyacrylamide, an inert ethanol precipitation aid), not a porous gel. Bis-acrylamide cross-links the acrylamide chains and this is what gives rise to the formation of the porous gel matrix. The amount of crosslinking, and therefore the pore size and consequent separation properties of the gel can be controlled by varying the ratio of acrylamide to bis-acrylamide. (Shi and Jackowski 1998).
TEMED (14.4 M) is responsible for initiating polymerization chains. The more TEMED you use, the shorter the resulting chains, and the more brittle the gel becomes. This creates difficulties in gel drying and in freezing gels for long exposures with film or imaging plates.
I routinely use 50 uL of 10% APS per 100 mL of gel.
Increasing the concentration of the initiators (ammonium persulfate and TEMED) results in a decrease in the average polymer chain length, an increase in gel turbidity, and a decrease in gel elasticity. Excess ammonium persulfate and TEMED have other effects, including oxidation of sample proteins (especially sulfhydryl-containing compounds) and changes in buffer pH. Excess TEMED can increase buffer pH, react with proteins (Dirksen and Chrambach 1972; Chrambach et al. 1976), and alter the banding pattern (Gelfi and Righetti 1981a). For more information on polyacrylamide gel polymerisation see Biorad Bulletin 1156.
I have developed a method for polymerizing gels that reduces the amount of TEMED as the gel concentration increases. All gels polymerize at the same rate, and even 20% acrylamide gels remain flexible and are easily dried down or frozen. Scale down the gel volume as required. I wait 45 - 60 minutes before using the gels.
Initiator concentration for degassed gel solutions
% acryl. for 100 mL gel 4% 6% 8% 10% 12% 14% 15% 16% 18% 20%
µL TEMED for native gel 24 22 20 18 16 14 13 12 9.5 7
µL TEMED for denaturing gel 22 20 18 16 14 12 11 10 7.5 5
thanks for setting us straight, tfitzwater. i never considered the ramifications of speeding up the polymerization process (shorter strands). makes a lot of sense. the method you give will be useful.
THANK YOU!! THIS IS GREAT!!