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Gel electrophoresis: samples running towards the middle?

Gel electrophoresis: samples running towards the middle? - Protocols and Methods Forum

Gel electrophoresis: samples running towards the middle? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 12-17-2003, 08:54 PM
Peter Frank
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Default Gel electrophoresis: samples running towards the middle?



Hello,

I do denaturing SDS polyacrylamide gel electrophoresis (SDS PAGE)
according to Laemmli (i.e. with a stacking gel and a separating gel)
for analytical separation of proteins. I use a vertical BioRad gel
electrophoresis apparatus.

1) One of my problems is that the samples tend to run towards the
middle, i.e. they do not run in a straight vertical line but rather
bent towards the middle. This effect is strongest for the outer lanes.

2) Another problem is that my bands become diffuse in the lower
molecular weight region although I use a 15 % SDS polyacrylamide gel.
The weight of proteins in that region ranges from about 18 kDa to 30
kDa. Literature says proteins of that size can still be separated by
SDS PAGE.

What could these effects be due to? What am I doing wrong? What can I
do to get prettier results in my SDS PAGE?

Peter
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  #2  
Old 12-18-2003, 10:40 AM
Kyle Legate
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Default Gel electrophoresis: samples running towards the middle?


"Peter Frank" <peter_frankde@yahoo.de> wrote in message
newsif1uv8mdh8325bv09p604i989qeb9lccb@4ax.com...
This problem usually arises when the samples contain different salt
concentrations. Equalize the concentration of salt in all samples and if you
are not using all the lanes, load the empty lanes with SDS-PAGE load buffer
containing the same salt concentration as your sample lanes.


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  #3  
Old 12-18-2003, 03:24 PM
Rene
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Default Gel electrophoresis: samples running towards the middle?

I regularly used a Laemmli system with additional urea, and good
flushing of the sample wells before loading was quite important in
order to avoid this problem. Besides, I did a prerun of about 10-15
min before loading the samples, although this is not recommended for
this (discontinue) system. Oh well, whatever works!

Rene.

"Kyle Legate" <[Only registered users see links. ]> wrote in message news:<a75c1648c1a3915cae66f4d2ba50bcbf@news.terane ws.com>...
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  #4  
Old 12-18-2003, 04:55 PM
Dr. Hiranya S. Roychowdhury
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Default Gel electrophoresis: samples running towards the middle?


Kyle is right. It is rather frustrating at times when the sample volumes
differ greatly due to the protein concentrations. I have, in my life so far,
run close to a thousand protein gels of different kinds and a few important
things to have a "pretty" picture that I have settled on are:

1. Very clean plates
2. Very clean, dust-free spacers (especially at the edge where they make
contact with the gel. If using alcohol to wipe the components, make sure no
trace of that is left.
3. As-close-to-equal volume loads as possible with low salt concentration.
Salt may be dialyzed out using microdialysis on a GVWP membrane (takes about 20
minutes)
4. Separating gel polymerization time should not be less than an hour.
5. The Stacking layer (low pH) should not be poured until one is ready with the
samples and is absolutely sure that the electrophorsis will begin within about
an hour!
6. The wells should be cleaned with the tank/running buffer using a syringe
prior to loading samples.
7. The buffer components used (including the running buffer) should be of good
quality.
8. Although most protocols talk about constant current, constant voltage works
much better in producing "pretty" pictures. The decreasing PD under constant
current causes the low Mr polypeptides to show up diffused. Also, the
voltage/current should be kept at the optimum.
9. The volume of the load should preferably be kept low. Thumb rule: the
height of the load less than half of the stacking gel height.
10. for a regular sized (20cm) 1.5mm gel, no more than 100ug of a total crude
extract protein/lane. For 0.75mm, it should be <50ug/lane. For partially
purified proteins, it is <50ug/lane and <20ug/lane. Then again, these values
are better impiricaly determined.


Quoting Kyle Legate <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---
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  #5  
Old 12-18-2003, 06:10 PM
Kyle Legate
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Default Gel electrophoresis: samples running towards the middle?

"Dr. Hiranya S. Roychowdhury" wrote:
I agree with all of these points, with two comments:

I have routinely poured 30 gels at a time in a multicaster and stored them
upright, combs intact, in a sealable plastic container with a thin layer of
water at the bottom, in the coldroom for 2-3 weeks at a time with no
noticeable decrease in quality.

I run Biorad mini gels (~5cm separating length) at 200 volts constant
voltage. It takes around 45 minutes for the dye to reach the bottom, and I
have never had a problem with fuzzy bands above, say, 6 kDa.


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  #6  
Old 12-18-2003, 07:34 PM
Peter Frank
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Default Gel electrophoresis: samples running towards the middle?

Thanks for the many useful tips!

Peter


Dr. Hiranya S. Roychowdhury wrote:

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  #7  
Old 12-18-2003, 09:10 PM
Dr. Hiranya S. Roychowdhury
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Default Gel electrophoresis: samples running towards the middle?

I haven't had such luck with even when I pourted the stacking gel the night
before.

And, I did mention that constant voltage is superior to constant current so far
as "crispy" separation is concerned. For me, mini gels (0.75mm) run in 45 min
at 150V :-)




Quoting Kyle Legate <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---
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  #8  
Old 12-19-2003, 10:59 AM
Dr Engelbert Buxbaum
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Default Gel electrophoresis: samples running towards the middle?

Peter Frank wrote:


1) This could be an effect of gel heating. Make sure that the current is
not too high (10-15 mA/gel should be all right), salt concentration in
the sample is ALARA and that you use the cooling loop of your apparatus.
Best cooling is achieved with a cooling water bath set to about 15
degrees C.

2) Try gradient gels for crisper bands. For a 18-30 kDa weight range I
would probably try a 10-15% T gradient. 5-15% if you need higher MW
proteins as well.
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  #9  
Old 12-19-2003, 08:40 PM
Tom Anderson
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Default Gel electrophoresis: samples running towards the middle?

On Fri, 19 Dec 2003, Dr Engelbert Buxbaum wrote:


The description certainly sounds like the phenomenon known as 'gel
smiling', which is indeed due to overheating.

tom

--
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL

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  #10  
Old 12-19-2003, 08:42 PM
Tom Anderson
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Default Gel electrophoresis: samples running towards the middle?

On Fri, 19 Dec 2003, Tom Anderson wrote:


Actually, now i read it again more closely, it doesn't at all.

Sorry.

tom

--
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL

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