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37kb and 7kb protein in western blot


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#16 mdfenko

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Posted 28 May 2015 - 04:40 AM

what is the formulation of the transfer buffer?

 

if it includes sds then you must include methanol. in fact, you should include methanol whenever transferring from sds-page to ensure stripping of the sds from the protein so that it won't interfere with binding to the membrane.


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#17 kegaff

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Posted 28 May 2015 - 07:57 AM

39 mM glycine, 48mM tris base, 0.025% sds, 20 % methanol pH 8.3. I even tried with one without sds. Both gave same result. I have another question. The boiled membrane after transfer showed more clear band than ur boiled one. But the only problem is boiled membrane is darker and the bands are lighter. The un boiled membrane has clear background but faint band. I cannot decide which blot to work on.i was wondering how can i get rid of dark background for the membrane thats boiled for 10 mins. Or maybe increase band intensity for un boiled membrane. Transfer time or voltage is not helping. The band is barely there. I even tried increasing the concentration of protein sample. The lane got too darker. I can only tell that the protein is there at 7 kb but it's very difficult to make it appear as a clear distinct band.

Edited by kegaff, 28 May 2015 - 04:26 PM.


#18 kegaff

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Posted 28 May 2015 - 11:34 AM

Mdfenko, could you plz send me the protocol of your low mw protein extraction along with the recipe of the extraction buffer and cocktail? I wanna start again. I am afraid what I am seeing is not the right band.

#19 mdfenko

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Posted 29 May 2015 - 04:04 AM

the addition of sds in the transfer buffer is generally to facilitate the transfer of high molecular weight proteins. it is usually not necessary for smaller proteins.

 

why and in what do you boil your membrane after transfer? i could understand it if you transferred from a non-denaturing gel and wanted to denature the protein with sds after transfer but you are using sds-page.

 

as for high background, you may not be blocking sufficiently or you are using the wrong blocking agent. also, are you boiling before or after blocking?

 

i have no protocol to give you. i worked with small proteins and peptides but wasn't extracting them. yours should be fine, just remember to remove the detergents after extraction.


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#20 kegaff

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Posted 29 May 2015 - 06:50 AM

After transfer, i keep the membrane in boiling water for 10 mins. This process is for fixation of small protein, i guess. I do my blocking at 5 % milk in TBST overnight at 4C. So is TCA precipitation enough to take off the detergents?

#21 mdfenko

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Posted 29 May 2015 - 07:12 AM

tca should be good for removing all of the buffer components. you can confirm this by adding tca to some buffer and determining if it gets turbid or if you can pellet a precipitate.

 

you're blocking with milk, are you using a biotinylated detection system? if so then you'll want to replace the milk, it contains biotin.

 

the proteins should fix to the membrane without having to boil.


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#22 kegaff

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Posted 29 May 2015 - 08:47 AM

One problem with tca precipitation is that i usually dissolve directly in tris tricine sample buffer directly. And store at -20c. When i am ready doing gel run, i take it out thaw and add equal volume of sds plus bME. Generally 5 microliter of protein sample and 5 microliter of sample buffer with Beta ME. And then load it. Since I see a huge pellet in the tube after precipitation i m hoping that I have enough protein in there. I generally start with 200 microliter of extracted orotein and dissolve in 40 microliter. The conc is 3.5 microgram per microliter so literally i am dissolving 700 microgram in 40 microliter. I maybe losing some while i am precipitating. I do this because i am not aware of any better sample solubilization buffer to do the Bradford again after tca precipitation. I found this solubilization buffer 8 m urea, 4 % chaps, 1% DTT but i afraid to use this. The tris trcine sds page protocol by schagger mentions of 3 times diluted sample buffer for dissolving but it has 12 % sds and even if we dilute it by 3 times , sds would still be more. The sample buffer for triis tricine from biorad has 2 % sds only. But as I said, after dissolving in sample buffer i cannot do Bradford.
So just wondering, am i messing up any step here? Or what could be a best solubilizing agent for low mw protein such that I can do Bradford after tca ppt also to know how much protein is going in?

#23 mdfenko

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Posted 01 June 2015 - 04:26 AM

i used to completely dissolve the tca pellet with sample buffer with sds and 2me. you may be able to more completely dissolve the pellet by homogenizing with a pestle or sonication (prior to addition of sds) then incubating at 60-70C for 10-20 minutes after addition of sds and 2me.

 

there are protein assays available that work with detergent and reducing agent present (sort of). look at this webpage from pierce. just don't add the tracking dye until after the assay.


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#24 kegaff

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Posted 03 June 2015 - 07:47 AM

When i did Bradford then i came to know that my tca precipitated protein has far less concentration of protein than i expect ed. Partly because all of the pellet is not dissolved in there. We do not have sonic action machine. I have to rely on solubilizing solution and it does not dissolve completely. I am too afraid to heat above 60 but its so hard to dissolve even at 60.

#25 mdfenko

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Posted 05 June 2015 - 04:03 AM

don't be afraid to heat above 60C in the presence of sds and 2me.

 

you can try a pestle like this one from fisher to disperse the pellet prior to incubation with sample buffer.


Edited by mdfenko, 05 June 2015 - 04:04 AM.

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