Basic question about electrophoresis
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Aaron Hicks
Mar 5, 2009, 4:21:44 PM3/5/09
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When I see statements about "X volts per cm," that refers to the length of the gel bridge between the two wells that contain platinum electrodes, right?
Why do many controllers go to higher voltages (several hundred VDC) if a few volts will suffice- does it provide greater speed, and does resolution suffer as voltage increases? I know there are heating effects at higher voltages.
Lastly, when it comes to gel electroimmunodiffusion, are the rules the same as with gel electrophoresis for proteins?
Believe it or not, I've been reading the books on it but a few of the basic tenets have escaped me!
-AJ
Why do many controllers go to higher voltages (several hundred VDC) if a few volts will suffice- does it provide greater speed, and does resolution suffer as voltage increases? I know there are heating effects at higher voltages.
Lastly, when it comes to gel electroimmunodiffusion, are the rules the same as with gel electrophoresis for proteins?
Believe it or not, I've been reading the books on it but a few of the basic tenets have escaped me!
-AJ
E. M. Muralidharan
Mar 5, 2009, 7:44:23 PM3/5/09
to diy...@googlegroups.com
I want to add a few more questions to those by Aaron. The books have not really been very helpful and the experts I asked, nonchalant.
Is the mechanism by which molecules move in the gels during electrophoresis fully understood? How do the molecules of different molecular weights, lengths, primary, secondary and tertiary configurations and charges move and get separated.
The gels are as varied as the molecules that are being separated in them. From paper to highly purified agarose and now agar, gelatin and what else.
What is the basis of using vertical and horizontal gels for the same class of molecules?
Like Aaron I am searching for the basic tenets.
Murali
Is the mechanism by which molecules move in the gels during electrophoresis fully understood? How do the molecules of different molecular weights, lengths, primary, secondary and tertiary configurations and charges move and get separated.
The gels are as varied as the molecules that are being separated in them. From paper to highly purified agarose and now agar, gelatin and what else.
What is the basis of using vertical and horizontal gels for the same class of molecules?
Like Aaron I am searching for the basic tenets.
Murali
Josh Perfetto
Mar 5, 2009, 7:50:24 PM3/5/09
to DIYBio Mailing List, Aaron Hicks
On 3/5/09 1:21 PM, "Aaron Hicks" <aaron...@gmail.com> wrote:
> When I see statements about "X volts per cm," that refers to the length of the
> gel bridge between the two wells that contain platinum electrodes, right?
This would be the distance between the positive and negative electrodes
> Why do many controllers go to higher voltages (several hundred VDC) if a few
> volts will suffice- does it provide greater speed, and does resolution suffer
> as voltage increases? I know there are heating effects at higher voltages.
Some people run gels that are much longer (around 30cm), some people use
power supplies to do protein gels or protein transfers which can require
much higher voltages, and yes you can increase the voltage to provide
greater speed at the expense of resolution. Increase it too much though and
the gel will melt, making it completely useless.
> Lastly, when it comes to gel electroimmunodiffusion, are the rules the same as
> with gel electrophoresis for proteins?
Some of the underlying principles are the same, but the methods/protocols
are quite different. In electroimmunodiffusion you are trying to quantify
the titer of a protein using a method that depends on antigen/antibody
binding. It's a more involved process than simply separating proteins by
size in a protein gel, though some of the underlying principles with gel
diffusion are the same. If you're interested in this, look up protocols on
electroimmunodiffusion.
-Josh
Eric Zhang
Mar 5, 2009, 7:54:56 PM3/5/09
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Murali,
This site may help you understand the basic tenets of DNA migration in agarose gels better:
http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html
-Eric
This site may help you understand the basic tenets of DNA migration in agarose gels better:
http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html
-Eric
Dan
Mar 5, 2009, 8:38:25 PM3/5/09
to DIYbio
Re: V/cm - this is simply a measure of how steep the voltage gradient
across the gel is. By rough approximation (the voltage drop per cm is
going to be different across the big buffer reservoirs than it is
across the gel), assume that a standard minigel box is 15 cm from
electrode to electrode. In order to get a 10 V/cm gel, you need 150
volts.
Generally, the trade off is that the faster you run your sample, the
more the gel heats. You can get weird striping and other artifacts if
you run a gel too hot. (That's not entirely a figure of speech - I
once melted a gel by running it at 300V to try and finish in time to
catch a bus)
As for the mechanism, this is all textbook stuff I read years ago so
who knows if its actually correct.
All gels are basically a molecular jungle gym filled with water. Any
molecule that tries to move through the gel has to navigate this
random network. Small molecules or molecules that are flexible and
linear can move more easily than large ones or long, inflexible ones.
Agarose gels have large pores since the agarose forms large
supramolecular assemblies, leading to a fairly open gel. Acrylamide
gives you a much tighter gel with small pores. While agar can be
used, I don't know how good it is - the charges in agar are much more
likely to interact in odd ways with the molecules you are
electrophoresing. Gelatin, paper and most other alternative means all
have various issues. There is a reason that labs use expensive
agarose for gels. As for the different agarose varieties, they tend
to be related to the melting point of the gel for various specialized
protocols.
Agarose is used for DNA gels since DNA is a relatively massive
molecule. Also, in the double stranded form usually run on gels, it
is a fairly inflexible molecule, limiting its ability to move through
gels. The open pores of agarose allow DNA to pass through in a
reasonable amount of time. Very small DNA molecules such as oligos
are often purified in acrylamide gels like proteins. DNA gels in
general are simple because the molecule has a consistent shape and
charge per MW. It is strongly negatively charged and moves towards
the positive electrode.
Acrylamide gels are used for proteins since they tend to be smaller
and require a finer gel to resolve them properly. Protein gels are
more complicated. Most protein gels are PAGE-SDS. PAGE stands for
the fact that it is an acrylamide gel (Poly-Acrylamide Gel
Electrophoresis). The SDS bit is for Sodium Dodecyl Sulfate. SDS is
a negatigely charged strong detergent that is very good at denaturing
proteins. The idea is that the SDS converts proteins from an array of
differently shaped, diferently charged things into fairly linear,
detergent coated molecules that are all negatively charged. In
theory, all PAGE-SDS protein gels should resolve proteins nicely by MW
since all the proteins are made linear and have a fairly consistent
amount of SDS bound to their hydrophobic regions. In practice, not
all proteins completely denature, SDS doesn't bind to proteins in a
linear fashion w/r to their MW and the original charge on the protein
all conspire to make gel-based protein mass measurements very dodgy.
Don't every assume that a protein MW from a gel is any better than
20%. Sometimes protein masses can be off by >100%.
There are non-denaturing gels that are basically PAGE-SDS gels minus
the SDS. In these cases, the molecular weight you get is pretty much
meaningless. You also have to be mindful of the protein's charge as
positively charged proteins will run right out of the wells backwards
unless you reverse the electric field from what's normal.
Vertical vs horizontal gels don't have anything to do with fundamental
differences in how the gels work. Instead, they are optimizations of
the various gel technologies and how to minimize the cost and maximize
the resolving power of the gels.
Agarose is relatively cheap (compared to polyacrylamide). The easiest
way to make a gel is to just pour it into a mold and have a comb cast
vertical wells in it. The wells are perpendicular to the gel run
direction since you want you DNA in as tight a band as possible. If
you had 'tall' wells, your eventual bands would also be tall and would
overlap and be diffuse.
Vertical polyacrylamide gels are limited by the fact that gel grade
acrylamide is very expensive. Here, you need a gel that's much
smaller in volume that's will capable of seperating large volumes of
proteins. In this case, you cast a gel between two sheets of glass.
The gel is vertical to aid in the pouring procedure. Unfortunately,
you have to make loading wells that are parallel to the gel run
direction to have enough volume to hold real world sample volumes.
This means that your bands would be unacceptably tall and diffuse. So
what's done here is the gel is poured in two parts - a resolving gel
and a separating gel. The wells and upper part of the gel are the
focusing region. The buffer pHs in the gel box buffer, protein
loading buffer, focusing and resolving gels are carefully chosen so
that the proteins in the wells is isoelectrically focused into nice,
tight bands in time for them to hit the separating buffer where the
proteins then separate from each other by MW like described before.
It's a complicated system that takes much more effort and cost to set
up than the agarose gels.
You can also run horizontal gels for proteins and vertical gels for
DNA. As mentioned before, very small DNA molecules are better run on
vertical polyacrylamide gels and vey large proteins can be run on
horizontal agarose gels. I'm sure there are other protocols where you
use an odd combination of sample and gel geometry.
There are tons of gel protocols such as pulsed-field gel
electrophoresis where you use AC currents to separate very large DNA
fragments. Comet DNA gels are where you embed cells in low melting
point agarose, lyse them and run out their chromosomal DNA to see if
there are many breaks in it from various environmental insults. I'm
sure there are plenty more that I'm forgetting or don't know about.
across the gel is. By rough approximation (the voltage drop per cm is
going to be different across the big buffer reservoirs than it is
across the gel), assume that a standard minigel box is 15 cm from
electrode to electrode. In order to get a 10 V/cm gel, you need 150
volts.
Generally, the trade off is that the faster you run your sample, the
more the gel heats. You can get weird striping and other artifacts if
you run a gel too hot. (That's not entirely a figure of speech - I
once melted a gel by running it at 300V to try and finish in time to
catch a bus)
As for the mechanism, this is all textbook stuff I read years ago so
who knows if its actually correct.
All gels are basically a molecular jungle gym filled with water. Any
molecule that tries to move through the gel has to navigate this
random network. Small molecules or molecules that are flexible and
linear can move more easily than large ones or long, inflexible ones.
Agarose gels have large pores since the agarose forms large
supramolecular assemblies, leading to a fairly open gel. Acrylamide
gives you a much tighter gel with small pores. While agar can be
used, I don't know how good it is - the charges in agar are much more
likely to interact in odd ways with the molecules you are
electrophoresing. Gelatin, paper and most other alternative means all
have various issues. There is a reason that labs use expensive
agarose for gels. As for the different agarose varieties, they tend
to be related to the melting point of the gel for various specialized
protocols.
Agarose is used for DNA gels since DNA is a relatively massive
molecule. Also, in the double stranded form usually run on gels, it
is a fairly inflexible molecule, limiting its ability to move through
gels. The open pores of agarose allow DNA to pass through in a
reasonable amount of time. Very small DNA molecules such as oligos
are often purified in acrylamide gels like proteins. DNA gels in
general are simple because the molecule has a consistent shape and
charge per MW. It is strongly negatively charged and moves towards
the positive electrode.
Acrylamide gels are used for proteins since they tend to be smaller
and require a finer gel to resolve them properly. Protein gels are
more complicated. Most protein gels are PAGE-SDS. PAGE stands for
the fact that it is an acrylamide gel (Poly-Acrylamide Gel
Electrophoresis). The SDS bit is for Sodium Dodecyl Sulfate. SDS is
a negatigely charged strong detergent that is very good at denaturing
proteins. The idea is that the SDS converts proteins from an array of
differently shaped, diferently charged things into fairly linear,
detergent coated molecules that are all negatively charged. In
theory, all PAGE-SDS protein gels should resolve proteins nicely by MW
since all the proteins are made linear and have a fairly consistent
amount of SDS bound to their hydrophobic regions. In practice, not
all proteins completely denature, SDS doesn't bind to proteins in a
linear fashion w/r to their MW and the original charge on the protein
all conspire to make gel-based protein mass measurements very dodgy.
Don't every assume that a protein MW from a gel is any better than
20%. Sometimes protein masses can be off by >100%.
There are non-denaturing gels that are basically PAGE-SDS gels minus
the SDS. In these cases, the molecular weight you get is pretty much
meaningless. You also have to be mindful of the protein's charge as
positively charged proteins will run right out of the wells backwards
unless you reverse the electric field from what's normal.
Vertical vs horizontal gels don't have anything to do with fundamental
differences in how the gels work. Instead, they are optimizations of
the various gel technologies and how to minimize the cost and maximize
the resolving power of the gels.
Agarose is relatively cheap (compared to polyacrylamide). The easiest
way to make a gel is to just pour it into a mold and have a comb cast
vertical wells in it. The wells are perpendicular to the gel run
direction since you want you DNA in as tight a band as possible. If
you had 'tall' wells, your eventual bands would also be tall and would
overlap and be diffuse.
Vertical polyacrylamide gels are limited by the fact that gel grade
acrylamide is very expensive. Here, you need a gel that's much
smaller in volume that's will capable of seperating large volumes of
proteins. In this case, you cast a gel between two sheets of glass.
The gel is vertical to aid in the pouring procedure. Unfortunately,
you have to make loading wells that are parallel to the gel run
direction to have enough volume to hold real world sample volumes.
This means that your bands would be unacceptably tall and diffuse. So
what's done here is the gel is poured in two parts - a resolving gel
and a separating gel. The wells and upper part of the gel are the
focusing region. The buffer pHs in the gel box buffer, protein
loading buffer, focusing and resolving gels are carefully chosen so
that the proteins in the wells is isoelectrically focused into nice,
tight bands in time for them to hit the separating buffer where the
proteins then separate from each other by MW like described before.
It's a complicated system that takes much more effort and cost to set
up than the agarose gels.
You can also run horizontal gels for proteins and vertical gels for
DNA. As mentioned before, very small DNA molecules are better run on
vertical polyacrylamide gels and vey large proteins can be run on
horizontal agarose gels. I'm sure there are other protocols where you
use an odd combination of sample and gel geometry.
There are tons of gel protocols such as pulsed-field gel
electrophoresis where you use AC currents to separate very large DNA
fragments. Comet DNA gels are where you embed cells in low melting
point agarose, lyse them and run out their chromosomal DNA to see if
there are many breaks in it from various environmental insults. I'm
sure there are plenty more that I'm forgetting or don't know about.
EJ
Mar 11, 2009, 12:11:54 PM3/11/09
to DIYbio
Very well said! Back when I poured my own gels, the "resolving" part
of the acrylamide gel did not have a separate buffer and was common
referred to as a "stacking gel". It was always a lower percentage
acrylamide than the separating gel.
of the acrylamide gel did not have a separate buffer and was common
referred to as a "stacking gel". It was always a lower percentage
acrylamide than the separating gel.
JonathanCline
Mar 13, 2009, 8:08:22 PM3/13/09
to DIYbio
On Mar 5, 7:44 pm, "E. M. Muralidharan" <emmur...@gmail.com> wrote:
>
> Is the mechanism by which molecules move in the gels during electrophoresis
> fully understood? How do the molecules of different molecular weights,
> lengths, primary, secondary and tertiary configurations and charges move
> and get separated.
Yes and no. DNA motion is so complex (long and stringy) that it is
>
> Is the mechanism by which molecules move in the gels during electrophoresis
> fully understood? How do the molecules of different molecular weights,
> lengths, primary, secondary and tertiary configurations and charges move
> and get separated.
still under active study regarding modeling the movement of the large
molecule through the matrix.
Check this journal for more info, although it's not open access, so
it's hard to get the articles without significant $$$.
Wiley Interscience, ELECTROPHORESIS
http://www3.interscience.wiley.com/journal/10008330/home
Improvements to separation methods is ongoing.. seemingly hampered
mainly by trying to manipulate nanotech systems (DNA, etc) without
having many nano-sized tools. Also, I'm guessing that a bunch of
electrophoresis methodology is set up because of the ways labs work
(process related issues) like having one person (lab tech's asst., for
example) make a batch of gels to be used later by the lab tech, which
to me explains the (what I view as inefficient) use of a comb for
making the wells -- vs. a hand tool which automatically makes the well
on demand and deposits the sample in one shot, eliminating loading
errors. It's a guess though, since I also hear plenty of, "I dunno,
that's just how we do it"'s. Maybe the biologists could complain
louder about the parts of wetwork that are so error-prone or time
consuming, then the engineers might be able to build something to fix
that (hence leaving biologists more time to actually do biology).
## Jonathan Cline
## jcl...@ieee.org
## Mobile: +1-805-617-0223
########################
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