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non denaturing SDS-PAGE

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cna_4reak
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Topic Started by cna_4reak
on 12/30/2009 20:42 PM   
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i am currently at a stage where i have purified my protein via ion exchange and gradient elution. so when i did SDS-PAGE, i found out there were few bands on the elution lane and the bands gradually faded. so the questions are:

1. if there were few bands observed, how do i determine which of the band(s) is my protein of interest?
2. i want to do native SDS-PAGE and localize the proteins straight away on the gel, but how do i concentrate the proteins without having to denature them?
3. is using PEG6000 better than acetone?
4. say if i sucessfully detect my protein band of interest, would silver staining the gel denature my native proteins? (coomasive staining doesn't work for me btw)



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Suola
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Posted By Suola
on 12/31/2009 7:30 AM   
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cna_4reak,

1. In non-denaturing electrophoresis, you need a migration standard for identification. You could load on another lane a pure protein sample, or compare pre- and post-induction aliquots in different lanes to see which band appears... This only works if your protein overexpresses well. You could also cut bands and do MALDI-MS experiment to see the molecular weight, or western analysis for definitive ID.

2. You can concentrate the protein with precipitation or spin-filter... google protein precipitation and you get lot of protocols. Here's a comparison of couple of methods.

3. It depends. There is no good algorithm to predict which method works for some particular protein. You need to try different methods and see which works the best. It also depends on the downstream application you want to do.

4. You can do silver staining and retain protein in native state, see here.

Could you elaborate why you want to do native page. You could assay the fractions by denaturing electrophoresis as well.

Cheers,

Certainty of death, small chance of success... What are we waiting for? - Gimli


Last edited Dec 31, 2009, 11:19 AM by Suola

cna_4reak
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Posted By cna_4reak
on 12/31/2009 19:49 PM   
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hi thx for ur reply

1. for migration standard, do you mean utilizing the protein marker (the one we usually load onto the gel) or do you mean a pure protein like BSA so that i know wether my technique for native SDS-PAGE is correct or not?

2. i choose to do native gel because i want to determine which of the protein band(s) would retain its enzyme activity. hypothesizely I would observed clear zones on that particular band(s).  but havent success on that part yet altough there are some journals that did it - so i assumed i might go for it.

3. at first i was thinking on slicing the bands on the gel and detect the enzyme activity from there onwards but i was afraid if the staining procedure might have any effects on the enzyme. are there any protocols in slicing the gel and getting the protein in its native state?




Suola
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Posted By Suola
on 1/1/2010 6:52 AM   
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cna_4reak,

May I ask why you are interested in the activity of some particular band? It would be easier to give helpful ideas if I knew what you are after. Are those bands fragments from a single protein? Or are you trying to pool "clean" fractions based on the activity in them?

The commercial mw markers are meant for denaturing PAGE only. Because the migration of native protein depends on both the charge state and hydrodynamic size, and the running buffer and some other things, the only standard that can be used in native page as migration standard is the very same protein you are purifying. So pure BSA wouldn't do either.

Cheers,

Certainty of death, small chance of success... What are we waiting for? - Gimli



cna_4reak
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Posted By cna_4reak
on 1/5/2010 1:59 AM   
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hi. sorry for the late reply. my project is basically purifying enzymes called azoreductase which is responsible in decolurizing synthetic dyes, which in this case i am utilizing reactive black 5 as my substrate for my enzyme.

below i have attached the result of my denaturing SDS-PAGE by silver stained. from the gel, there were two bands observed on lane e3, e4 and e5. so im wondering:

1. why do i get a few bands after it has underwent ion exchange chromatography?

2. if i considered the bands that appeared on lane e3, e4, and e5 as my purified enzyme, therefore which of those bands that actually work for my enzyme assay? thats why i was thinking of doing native SDS-PAGE. or are there any other way that i can do to detect enzyme activity from the gel?





Attached file: RESULT.pdf (4 downloads, 280KB)


Suola
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Posted By Suola
on 1/5/2010 8:24 AM   
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cna_4reak,

1. It is practically expected to have more than one band after single step of purification from a complex sample. They might be two proteins (glycoforms maybe) that have similar charge properties or a stable heterodimer complex, or just two unrelated proteins that need hight salt to elute.

2. If a colorimetric (or precipitation or other) assay exists, you can try to do it directly on a native gel without silver staining and compare the position of that signal to the bands on a silver stained native gel. The best way is to run a single gel with same sample on two different lanes, cut the gel in two and do the enzyme assay on the other half and silver stain on the other half. Compare the signal positions.

Have you done literature search on your protein? Is it a monomer, dimer?

Another option is to add second chromatography step and see if you get those protein separated.

Cheers,

Certainty of death, small chance of success... What are we waiting for? - Gimli


Last edited Jan 05, 2010, 10:48 AM by Suola

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