9 replies to this topic

[tamarine]

Hi, everybody.

I just wondering, anyone would help me with my IEF, such as : tips and tricks, stages, chemical reagent, and visualization. the tutorial and educational link would be really appreciated.

Please.., i am definitely desperate about this.

[Prep!]

hi tamarine.. i sould suggest the best bet is to call the technical persopn from Amersham (u say multiphor) for a demonstration. They will give a hands on demonstration with your consumables and reagents and samples. so the practicality of using the system shud not be a problem..
then once u start using the system.. the troubleshooting can be shared here so that we can help u out..

Edited by Prep!, 17 July 2011 - 09:06 PM.

[mdfenko]

do you have the book that came with the multiphor?

if not then here it is:

 multiphor II manual.pdf   1.69MB   854 downloads

if you still have questions then ask and try to be specific, we don't want to rewrite the manual.

[tamarine]

mdfenko, on 18 July 2011 - 12:02 PM, said:

do you have the book that came with the multiphor?

if not then here it is:

multiphor II manual.pdf

if you still have questions then ask and try to be specific, we don't want to rewrite the manual.

Hi, mdfenko.
I am so grateful you response my problem. Actually, the pdf file that you've attach me is similar with mine. i have some papers about it too. the technical person from the factory also have been gave me the demonstration. but, seems she is not quite understand well about the instrument it self.

What do you prefer of buying the precast (such as : Immobiline Drystrip gel) or made in laboratory? in working IEF with this instrument?
Same as gel, what about the IPG buffer?
Did it really able to reach 1000 voltage in separation (the recommendation mentioned), while i employ 300 volt did tear the gel?

This technique and its optimizing instrument were really challenging me. Your recommendation is highly appreciated.

[Inmost sun]

like for PAGE, you can choose between mini, midi and maxi IEF preparation; what do you need?

[mdfenko]

i used to use the original multiphor. the only major differences are that the electrodes are not adjustable and the cooling plate is narrower.

i cast my own and used precast, following the running recommendations for each and they all worked as advertised.

the power supply settings are limits. you may or may not approach a particular limit, it depends on the gel, temperature, humidity, etc.

the most important limit, and the one that it should actually run at for most of the separation, is power (watts). this will help prevent the gel from overheating, drying out in spots and bursting into flames (yes, i saw a gel start to flame in a spot, once).

[tamarine]

mdfenko, on 20 July 2011 - 12:20 PM, said:

i used to use the original multiphor. the only major differences are that the electrodes are not adjustable and the cooling plate is narrower.

i cast my own and used precast, following the running recommendations for each and they all worked as advertised.

the power supply settings are limits. you may or may not approach a particular limit, it depends on the gel, temperature, humidity, etc.

the most important limit, and the one that it should actually run at for most of the separation, is power (watts). this will help prevent the gel from overheating, drying out in spots and bursting into flames (yes, i saw a gel start to flame in a spot, once).

Thank you for your response..
How do you cast your gel?  Do you utilize the peristaltic pump to form the gradient pH gel? What kind of pharmalyte do you use? Would you guide me as well as you work with it? (really begging for this point so bad)
If you use the precast, it is quite expensive for daily work,right? So, what is your suggestion for the newbie like me.

If you employ the precast, how long do you need to separate the samples?
Do you use the strip buffer or precast buffer?

Pardon me, if I got you abundant questions. Your advise, really highly appreciated..

Thank you

[mdfenko]

tamarine, on 20 July 2011 - 08:56 PM, said:

How do you cast your gel?  Do you utilize the peristaltic pump to form the gradient pH gel? What kind of pharmalyte do you use? Would you guide me as well as you work with it? (really begging for this point so bad)
If you use the precast, it is quite expensive for daily work,right? So, what is your suggestion for the newbie like me.

If you employ the precast, how long do you need to separate the samples?
Do you use the strip buffer or precast buffer?

it's been a long time since i last ran the multiphor so i may not be able to answer all of your questions but most of the answers are in the literature that came with the apparatus and the precast gels.

when i poured my own i was not pouring gradients, you do that if you are pouring an immobilized pH gradient (using immobilines, as with a dry strip). if you are using pharmalytes (or any other brand ampholytes) then you just pour the gel solution into the sandwich, seal and allow to polymerize. we used a binding film, in the sandwich, to serve as a gel support for the separation.

precast gels are not as expensive, per sample, as you may think and they should give consistent results (we started with precast then moved on to pouring our own). we liked the serva thin precast gels as well as the "normal" thickness gels sold by ge healthcare (amersham, pharmacia, lkb). we used the filter paper buffer strips and "buffers" (in quotes because some of them aren't actually buffers) called for in the procedure sheets that came with the gels.

as for which ampholytes (or blend of ampholytes) to use, that depends on the pH range that you require for your purpose.

[tamarine]

mdfenko, on 21 July 2011 - 07:39 AM, said:

tamarine, on 20 July 2011 - 08:56 PM, said:

How do you cast your gel?  Do you utilize the peristaltic pump to form the gradient pH gel? What kind of pharmalyte do you use? Would you guide me as well as you work with it? (really begging for this point so bad)
If you use the precast, it is quite expensive for daily work,right? So, what is your suggestion for the newbie like me.

If you employ the precast, how long do you need to separate the samples?
Do you use the strip buffer or precast buffer?

it's been a long time since i last ran the multiphor so i may not be able to answer all of your questions but most of the answers are in the literature that came with the apparatus and the precast gels.

when i poured my own i was not pouring gradients, you do that if you are pouring an immobilized pH gradient (using immobilines, as with a dry strip). if you are using pharmalytes (or any other brand ampholytes) then you just pour the gel solution into the sandwich, seal and allow to polymerize. we used a binding film, in the sandwich, to serve as a gel support for the separation.

precast gels are not as expensive, per sample, as you may think and they should give consistent results (we started with precast then moved on to pouring our own). we liked the serva thin precast gels as well as the "normal" thickness gels sold by ge healthcare (amersham, pharmacia, lkb). we used the filter paper buffer strips and "buffers" (in quotes because some of them aren't actually buffers) called for in the procedure sheets that came with the gels.

as for which ampholytes (or blend of ampholytes) to use, that depends on the pH range that you require for your purpose.

Absolutely, i am grateful for your guidance. it quite help me with the problems. Now, could you give a favor?
What brand of precast gel you've used? so, i can purchase my own.
For, the first time like this. should i use the broad range of pH gradient? and later on, i can use the narrower which is appropriate with my sample?
i have ever heard that the pieces papers of sample (amersham) and spread all around the gel is good enough to test the optimization of pH gradient. is it so?

Thank you very much.

[mdfenko]

tamarine, on 23 July 2011 - 03:14 AM, said:

What brand of precast gel you've used? so, i can purchase my own.
For, the first time like this. should i use the broad range of pH gradient? and later on, i can use the narrower which is appropriate with my sample?
i have ever heard that the pieces papers of sample (amersham) and spread all around the gel is good enough to test the optimization of pH gradient. is it so?

i mostly used servalyte precotes.

i also used ampholine pagplates by ge healthcare.

i would recommend using broad range, pH 3-10, for your initial runs.

the paper piece sample applicators work well but i prefer using the sample application strips.