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Native PAGE Problems in Protein Migration

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VinBio
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Topic Started by VinBio
on 3/23/2009 0:39 AM   
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hii all, Please kindly help me in this...

when i run Native PAGE with my crude protein extract ..the protein of my interest doesnt migrate down to anode & its accumulated at the well.

i use stacking gel 1M Tris 6.8 pH & resolving gel is 1.5M Tris pH 8.8, Acrlyamide/Bisacrlyamide  29+1 ,

Running buffer is 1X Tris glycine pH 8.3, loading buffer is 1X tris pH 6.8 with glycerol n BPB.
i also flush the wells b4 loading


pI of my protein sequnce (as calculated by pi calculator software) is 5.7 & im using K phosphate buffer pH 7.0 while extracting it from cells.

The funny thing is... after comassie staining d gel... i get bands but without my protein.... banding pattern in control n test is the same!!

However my protein is showing good activity in assays in same buffer (KPO4 pH 7.0) ...

I came to know that my protein is at the wells by doing a On gel assay after running the native.


Its just a 52kd protein which is non globular, non fibrial....     I also tried pre-running the gel for 20mins but it didnt help...the protein is just not ready to come down

I dont know wats the problem!... Plz help me.. if anyone experienced this problm b4...

I have rechecked all my buffers pH & there is no problem with them..

Also to mention.. im using crude periplasmic extract,  the supernatent is clear...




I will be really grateful for your replys ..im worried about this as i cannot move further without a purified protein....


Last edited Jan 25, 2010, 10:19 AM by varsha
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varsha
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Posted By varsha
on 3/23/2009 11:23 AM   
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My first guess is that the protein in question is present in a multimeric (hetero and less likely homo) complex in your crude extract. The supernatant from your prep could have complexes of proteins hundreds/thousands of kilodaltons in size. Good nw is that you have activity in your prep.


You may be able to  fractionate your protein complexesby running your supernatant through size exclusion chromatogrphy and assaying each fraction for activity You may want to start with S200 from GE (Amersham). Alternatively, if you do not have access to  HPLC columns, you could run your supernatant on a 5-15% sucrose/glycerol ultracentrifugation gradient and, again, assay each fraction for activity. Either by HPLC or ultracentrifugation, you would get some idea of the size of complex your protein is present in.


I would also look into known or predicted interactions for your proteins. You could IP you protein from the fraction or do mass spec to identify other proteins in the complex.


Good luck!



VinBio
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Posted By VinBio
on 3/24/2009 22:56 PM   
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Thanks for the guidunce Varsha..


So in a crude extract, it it possible that a 52kd small protein be complexed with other unrelated protein eg. membrane proteins or fragments as it was isolated from periplasmic membrane?


Yes I can carry out Ultracentrifugation as we dont have HPLC in our own premises. But i have to use glycerol rather that sucrose.. As sucrose is the substrate for my enzyme...


I'll reply soon regarding the results...



VinBio
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Posted By VinBio
on 3/25/2009 7:30 AM   
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Hi again,


One more doudt I had..


when i run SDS PAGE of the protein extract, I get same banding pattern in control & test ..


DO u think it is because of the protein being complexed in a crude extract with other proteins fragments?


As my gene insert is not in a overexpressing vector..it can be possible that my protein is produced in very less amount which is not clearly detected in SDS but is in sufficient amt. to give activity...


wat do u think?



ARGERINE
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Posted By ARGERINE
on 4/4/2009 1:22 AM   
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I will recommend running a native PAGE and SDS PAGE together...


run yr SDS PAGE gel as usual but the native PAGE with a running buffer using TRICINE instead of glycine and make the pH of bufffer 9.2. as well as of yr resolving gel too.


Make yr gel 12%T and 2.8%C


Load yr samples in naitve page as follows


crude extract


crude extract in 8M urea


crude extract reduced  with DTT and alkylated with Iodoacetamide.


and finally the crude extract with SDS sample buffer ( remember its native PAGE only yr sample is in denturing conditions)


This will tell you all the possibilities that are occurrung over there If yr protein is monomeric or multimeric and chains how many.....????? if you have .......


Do let me know I may help you more with it.


once you get the results


 



Oh,  one more possibiltiy


Just in case none of this works and your protein still remains in the gel thta means you have not been able to get through the charges on the protein and some how it remains uncharged and hence doesnt migrate with the pH you have provided it.

 In that scenario you RUN a CTAB NATIVE PAGE and you will see your protein migrating.


CHEERS

HAPPY PROTEIN HANDLING


Gaganjot

Gaganjot Singh

Truth seems so closer now......


Last edited Apr 09, 2009, 13:34 PM by frasermoss

VinBio
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Posted By VinBio
on 4/5/2009 7:47 AM   
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Thanks a lot Gaganjot for your reply


right now we are not having tricine with us..so have tocarry out this using glycine itself...


May i know wats the reason for using Tricine. & also urea ?


I thought of another possibility, suppose the calculated pI (which is 5.7) is incorrect & my protein is Basic in nature


Then it wont migrate in this present gel system..i guess in that case, i may have to use Acetic acid Urea electrophoresis. (Although there r no reports of the protein being basic in nature)


Right now the lab is not having material to perform IEF, esle i would have done that first.


The possibility of it being a basic protein seems very dense as when i perform SDS page, where the protein is denatured...there also i get same banding pattern in control & test !


& as i had mentioned the enzyme shows very good activity in phosphate buffer pH 7.0


I have never worked much with proteins, but is that a valid interpretation?


Also, Can you give me details about CTAB Native gel, i have never done tht before, can u give me the references /protocols for performing that... ? Can i use CTAB if the protein seems to be basic in nature?


Awaiting your reply..


Regards...


 


Last edited Apr 16, 2009, 9:35 AM by varsha

ARGERINE
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Posted By ARGERINE
on 4/5/2009 23:25 PM   
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tricine has a higher pKa then glycine and it has 3 charges compared to 1 of glycine so it has a ways too better resolution comapred to glycine PAGE.



If you are somewhere closer to Delhi, India. I can help you with tricine stocks and PAGE. I have been running electrophoresis all my life in research and i hope there is virtually no problem that cant be overcome by just buffers.


you can try glycine PAGE only but use your resolving gel 12% an at pH 9.2 instead of normal 8.8. and resolving buffer also has a pH of 9.2.

Bands wouldnt be that sharp as in tricine page but resolution will be somewhat similiar.


Urea will denature the protein completely so its useful to use urea PAGE


I will soon post the protocol for CTAB PAGE. CTAB is a cationic detergent and the principle is just the same as that of SDS PAGE except for  the point that migration is in opposite direction because sds is anionic and CTAB is cationic.


cheers.

Gaganjot Singh


Gaganjot Singh

Truth seems so closer now......


Last edited Apr 09, 2009, 13:33 PM by frasermoss

VinBio
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Posted By VinBio
on 4/7/2009 23:24 PM   
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Thanks for your reply Gaganjot,


What do you think about the nature of the protein?  ..It indicates to be a basic in nature.. although i have to confirm that..


also, may i know the conc. of DTT & indoacetamide to be added while loading?


Is it fine to use indoacetic acid instead of iodoacetamide?.. as its also an alkylating agent...


i couldnt find your mail id,  may i get your mail id plz?


my mail id :  vinitivaidya@gmail.com


 


 


Last edited Apr 16, 2009, 9:41 AM by varsha

ARGERINE
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Posted By ARGERINE
on 4/8/2009 0:00 AM   
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USE 100mM OF IODOACETAMIDE AND 50mM OF DTT


ARGERINE@GMAIL.COM.


SEND ME THE DETAILS.....


GAGANJOT SINGH

Gaganjot Singh

Truth seems so closer now......


Last edited Apr 16, 2009, 9:33 AM by varsha

ARGERINE
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Posted By ARGERINE
on 4/16/2009 2:47 AM   
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I surely will try and send. The only problem is courier's are security checked and may be they wont allow sending some unknown falcons with chemicals...... I have a busy schedule till this monday .... and then tuesday I will send on the address you mentioned in your email.

Gaganjot Singh

Truth seems so closer now......



VinBio
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Posted By VinBio
on 4/17/2009 23:40 PM   
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Thanks a lot Gaganjot....


Regards



nanobio
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Posted By nanobio
on 6/25/2009 10:51 AM   
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hi

i run native PAGE  for bacterial protein and i get more than one band instead of getting single band. what could be the reason. is there any special protein extraction procedure in order to run in native PAGE. please help.

thanx,  Gaganjot Singh for suggesting me to  run native PAGE  instead of  SDS-PAGE to raise antibodies!


Last edited Jun 25, 2009, 12:56 PM by nanobio

msvnathan
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Posted By msvnathan
on 6/25/2009 12:17 PM   
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hi my reply to nanobio : 6/25/2009 10:51 AM Is your staining procedure is specific to that protein in Native Gel? if so surely you will get single band.



nanobio
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Posted By nanobio
on 6/26/2009 20:08 PM   
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i stained it with coomasie blue as we do with SDS PAGE. i use protein from Erwinia spp. mixed with bromophenol blue dye for loading. Is there any specific dye to stain the native gel?

thanks very much for your answer.


Last edited Jun 26, 2009, 22:19 PM by nanobio

dcbayer04
China

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Posted By dcbayer04
on 11/6/2009 5:11 AM   
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Hi all,

I was experiencing almost the same problem as VinBio. The only difference may be that my interested protein was at the starting point of the Native PAGE gel rather than at th wells, which was reflected by in-gel activity test.

So according to varsh and Gaganjot's suggestion, the reason may be the nding of the protein with the other unrelated proteins to form multimeric complex, or because of the unsuccessful charge of the protein. I guess the second may not be the reason because my protein actually moved from the well to the interface of the stacking and running gel, am i right? Any other possibilities? Is it going to work if I cut the desired part of gel and elude for a second SDS-PAGE?

Regards

Ding Chang



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