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SDS PAGE- HELP


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#1 Arunkp

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Posted 14 October 2010 - 08:30 PM

Hi to All,

I have started doing work with SDS PAGE,

I could make the gels (Stacking and Resolving- as per the manual - 5% and 12% respect.)

But many time, I could see my bands struck in the region where Stacking and separating joins(interface)

Also, some times Bands moved but they are not clear and could not able to see it properly,

I have tried running only Separating gel alone, but same happens BNADS ARE NOT VISIBLE.

A fresh Staining and destaining has been used everytime, Samples in sample buffer were boiled till 90 C for half hour, fixing done soon after removal from Slab-20% TCA,

I dont know whats happening,

10ul of Medium range Marker was used and atleast 200ul of Sample buffer was added and boiled - but no bands AtAll.

See the pic added

Kindly help me out for this...

Arun

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#2 ElHo

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Posted 15 October 2010 - 02:34 AM

Do you have to boil your samples for half an hour? This is usually done for only 5-10 minutes. Extended boiling may cause aggregation, especially of larger proteins, and the proteins will not enter the separating gel. Are you sure you also have to add sample buffer to your marker and boil it? Because our protein standards are all ready to use. In my opinion there are some detectable bands in your left lane, although they are quite diffuse. What does the standard u use in your lab look like after staining when one of your colleagues doing this?

#3 K.B.

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Posted 15 October 2010 - 06:37 AM

If the MW marker is not visible there must be something very wrong with the electrophoresis. The two usual suspects are the procedure (wrong proportions, wrong mix etc) or the reagents (eg. old, wrong pH etc.) Please, describe your procedure in more details - composition of solutions you use, what do you mix and in what proportions etc. and check dates of preparation and pH of your reagents (ie. buffers you use for making gel and running buffer).

I agree with ElHo - 30 minutes of boiling is too much. I use 5 minutes at ~95 degrees C (ie. very hot but not boiling).

As for staining - I use and re-use "home made" colloidal Coomassie without any problems. I had a 500 mL bottle of colloidal Coomassie that was technically "used" but worked very fine for at last two years. :)

#4 GNANA

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Posted 15 October 2010 - 09:09 AM

check the pH of ur resolving gel buffer. 30 mins boiling is too much, i usually boil at 99 for 8 mins,,,make sure to load atleast 20 microgram of protein per well.

Edited by GNANA, 15 October 2010 - 09:15 AM.

I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#5 shivasankari

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Posted 23 November 2010 - 03:09 AM

Hi to All,

I have started doing work with SDS PAGE,

I could make the gels (Stacking and Resolving- as per the manual - 5% and 12% respect.)

But many time, I could see my bands struck in the region where Stacking and separating joins(interface)

Also, some times Bands moved but they are not clear and could not able to see it properly,

I have tried running only Separating gel alone, but same happens BNADS ARE NOT VISIBLE.

A fresh Staining and destaining has been used everytime, Samples in sample buffer were boiled till 90 C for half hour, fixing done soon after removal from Slab-20% TCA,

I dont know whats happening,

10ul of Medium range Marker was used and atleast 200ul of Sample buffer was added and boiled - but no bands AtAll.

See the pic added

Kindly help me out for this...

Arun


Hi Arun,
I second the above saying - no need to heat the sample for 30 min. I generally heat at 95 deg for 5 min and if the sample contains cells then for 10 min.
And with your first problem, on your protein struck in between, I would suggest you to completely dry the ethanol/water that you would have poured on the top of the sepa' layer. when there are traces of water/ethanol, polymerisation doesnt happen there and you have a gap in between. With regard to no protein bands, check you gel components, pH and also check your run, if you run for too long your protein runs into the buffer. And also check if your stains are good, if not you will have a band but might not be visible because of the problem in stain. And also run your gel at a lower voltage so as to avoid over heating of the gel.

cheers

Shiva
Cheers

Shiva




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