Electrophoresis Troubleshooting Forum

Electrophoresis Troubleshooting Forum http://www.protocol-online.org/forums Sun, 19 Feb 2012 01:19:38 +0000 1000 how to interpret data results of Bamh1 and SmaI? http://www.protocol-online.org/forums/topic/24484-how-to-interpret-data-results-of-bamh1-and-smai/ thank you.]]> Sun, 19 Feb 2012 01:19:38 +0000 http://www.protocol-online.org/forums/topic/24484-how-to-interpret-data-results-of-bamh1-and-smai/ Is that a Plasmid??? http://www.protocol-online.org/forums/topic/24445-is-that-a-plasmid/ Attached Image: 14 feb 1.jpg

]]> Wed, 15 Feb 2012 16:38:00 +0000 http://www.protocol-online.org/forums/topic/24445-is-that-a-plasmid/ <![CDATA[Is there an optimum % agarose or... "more is better"?]]> http://www.protocol-online.org/forums/topic/24211-is-there-an-optimum-agarose-or-more-is-better/
Thank you.]]> Mon, 30 Jan 2012 17:54:32 +0000 http://www.protocol-online.org/forums/topic/24211-is-there-an-optimum-agarose-or-more-is-better/ 2 D electrophoresis _ proteins do not move to lower part of analytic gel http://www.protocol-online.org/forums/topic/24166-2-d-electrophoresis-proteins-do-not-move-to-lower-part-of-analytic-gel/ Hi,
We have a huge problem with a second dimension (SDS-Page) during 2-D
electrophoresis protocol. After isoelectric focusing, during SDS-Page, proteins leave stacking ("upper") gel, move fluently to analytic
("lower") gel and then do not move to lower part of analytic gel. When proteins should move from upper part to
lower one, they are "blocked" and after that, only pigment
(bromophenol blue) moves down. We've changed all reagents, pH of
buffers is proper. We've tried to use three different ACs and three
different equipments for electrophoresis. Electrode buffer is prepared
properly too. Second dimension electrophoresis runs on constant
intensity, but we've observed that volts are rising very fast.

Of course, it is not our first attempt. Before we don't have any
problems like that.]]> Thu, 26 Jan 2012 20:29:20 +0000 http://www.protocol-online.org/forums/topic/24166-2-d-electrophoresis-proteins-do-not-move-to-lower-part-of-analytic-gel/ Western Blotting Resolving Problem http://www.protocol-online.org/forums/topic/24025-western-blotting-resolving-problem/ I have some problem with my recent Western Blotting. I am checking for 180kda protein and so i run 8% gel. But for proper resolving I run the gel at 120V for 3.30 hrs (usual timing 2.30 hrs). I am able to see the band of my interest very clearly but not at 180 kda. Its near 140 kDa. If we overrun the gel is there any chances that our protein moves fastly than the marker. Kindly write your experience on this. Thanks in advance..]]> Mon, 16 Jan 2012 02:56:14 +0000 http://www.protocol-online.org/forums/topic/24025-western-blotting-resolving-problem/ RNA electrophoresis http://www.protocol-online.org/forums/topic/23798-rna-electrophoresis/
I will be really gratefull if someone can help me.

Sorry about my english.

Carola. Posted Image

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]]> Fri, 16 Dec 2011 23:52:49 +0000 http://www.protocol-online.org/forums/topic/23798-rna-electrophoresis/ precipitates in 50X TAE http://www.protocol-online.org/forums/topic/23442-precipitates-in-50x-tae/
i recently made 50X tae buffer and i notice some precipitates. is it normal??

many thanks.

S]]> Wed, 16 Nov 2011 16:02:48 +0000 http://www.protocol-online.org/forums/topic/23442-precipitates-in-50x-tae/ SDS-PAGE running conditions http://www.protocol-online.org/forums/topic/23405-sds-page-running-conditions/
I'm in a bit of a dilemma currently.

I want to push my proteins as tightly as i can within my stack to the resolving without much spread.
On one hand i can run it at 150V for 5-10mins to get everything through the stack and into the resolving
and on the other hand i can run it at 30V for 2-3hours to get the same result as above.

These would then be cranked up/down to say 60-90V to allow for the proteins to run through the resolving gel.

My question is whether the slower method is better than the faster method, or if anyone has any other ideas to get nice tight bands.

(These are all being run at 4degC)

Cheers.]]> Mon, 14 Nov 2011 10:30:54 +0000 http://www.protocol-online.org/forums/topic/23405-sds-page-running-conditions/ SB buffer and agarose: Fragments less than 100 bp http://www.protocol-online.org/forums/topic/23355-sb-buffer-and-agarose-fragments-less-than-100-bp/
I have a problem with a SNP, my genotyping bands are 70 and 40 bp. Try running in polyacrylamide, but I get a different pattern of restriction than expected. The person whopublishes recommended me a 2% agarose and buffer SB, but I have some doubts, bandsare going to see if they are so small? At what voltage I can run it? and for how long? Does the gel gets hot enough to run it cold? Bromide staining Does not give any problem?
Thanks for your answers]]> Mon, 07 Nov 2011 17:15:39 +0000 http://www.protocol-online.org/forums/topic/23355-sb-buffer-and-agarose-fragments-less-than-100-bp/ My protein samples are running abnormally on a SDS PAGE http://www.protocol-online.org/forums/topic/23354-my-protein-samples-are-running-abnormally-on-a-sds-page/ I have a peculiar problem in sample running on a SDS PAGE. We use biorad 's mini protean gel apparatus for casting and running the gel. The running was really abnormal for the last two or three gels, the samples in the wells had a "V" shape as it was running, bulging of samples was also seen. I never had such a problem. Please see the images and give your comments. After running the gel I have proceeded further to western blotting which was also a failure.

P.S: Zoom out the second image for clarity.

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