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kegaff's Content

There have been 14 items by kegaff (Search limited from 30-May 19)


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#175186 37kb and 7kb protein in western blot

Posted by kegaff on 03 June 2015 - 07:47 AM in SDS-PAGE and Western Blotting

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.



#175132 37kb and 7kb protein in western blot

Posted by kegaff on 29 May 2015 - 08:47 AM in SDS-PAGE and Western Blotting

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?



#175130 37kb and 7kb protein in western blot

Posted by kegaff on 29 May 2015 - 06:50 AM in SDS-PAGE and Western Blotting

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?



#175118 37kb and 7kb protein in western blot

Posted by kegaff on 28 May 2015 - 11:34 AM in SDS-PAGE and Western Blotting

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.



#175115 37kb and 7kb protein in western blot

Posted by kegaff on 28 May 2015 - 07:57 AM in SDS-PAGE and Western Blotting

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.



#175100 37kb and 7kb protein in western blot

Posted by kegaff on 27 May 2015 - 09:56 AM in SDS-PAGE and Western Blotting

Omg i cannot believe, i saw the band at 7 kb. The membrane is little darker but i can see the band there. Now i just need to tweak to make it better. And the other challenging thing now is showing both the band in the same gel. 37 kb band is missing i was i did the transfer for 15 mins only at 13v. I will play around with volt to figure out the median transfer time and voltage for both the bands. Thank you all for all the comments and suggestion. I ll keep you posted.



#175096 37kb and 7kb protein in western blot

Posted by kegaff on 27 May 2015 - 05:09 AM in SDS-PAGE and Western Blotting

Thanks for your advice. I ll try that way.



#175089 37kb and 7kb protein in western blot

Posted by kegaff on 26 May 2015 - 10:07 PM in SDS-PAGE and Western Blotting

Thanks for your reply. SO what is your recommendation of a buffer? Should i decrease the concentration of sds? Or remove triton from the buffer?



#175061 37kb and 7kb protein in western blot

Posted by kegaff on 25 May 2015 - 01:00 PM in SDS-PAGE and Western Blotting

Oh i have question about sample concentration also. I am loading about 30 microgram of total protein. Do i have to load more for 6 kb protein detection?



#175060 37kb and 7kb protein in western blot

Posted by kegaff on 25 May 2015 - 12:56 PM in SDS-PAGE and Western Blotting

We use 1.5 volume protein extraction buffer( 100 mM tris HCl ph8, 1 mM EDTA, 150mM nacl, 1 % triton x) 50 microliter/ gm protease inhibitor cocktail, 10 mM DTT, 1 % chaps.
After extraction, when i did tca precipitation, i washed with acetone and directly added tris trcine sample buffer from biorad. The protein sample was still blue and not yellow so i didn't do any neutralization. However, it was too difficult to re suspend the protein in sample buffer so i incubated at 37 for half an hour. The easily dissolved low mw protein was directly loaded even though there was undissolved protein in the tube.



#175018 37kb and 7kb protein in western blot

Posted by kegaff on 23 May 2015 - 07:29 AM in SDS-PAGE and Western Blotting

Update- i got 37kb band only and not the 7 kb band. The front was so smeary with non tca ppt sample in tris tricin precast gel. I used the buffer also from biorad. Is it a transfer issue? Or is there a seperate protocol for getting low mw protein from filamentous fungi? Please help me if anybidy knows what went wrong.



#175005 37kb and 7kb protein in western blot

Posted by kegaff on 22 May 2015 - 09:37 AM in SDS-PAGE and Western Blotting

Thanks for your valuable comment.
So currently i am using 10-20% tris tricine buffer from biorad and sample buffer and tris tricine running buffer also from biorad with protein marker that can show even 1 kb band.
I did a semi dry transfer at 10v for 40 mins. Its a gradient gel, so i was not too much worried about that longer transfer. I could see 2kb ladder band too in my membrane. I haven't stained the membrane though. However, i am staining the gel and it looks pretty clear with less residue in there.
I am doing imaging in few hours 3-4 hours. Let's see how it turns out. But i have few questions regarding my protein sample.

I had 2 sets of protein sample. For half i did, no treatment for the other half i did TCA precipitation. When I was running the gel, the sample front moved down for tca precipitated ones while the other without precipitation did not actually moved away as the front. It made a kind of floral patch at 10 kb area. So i am thinking that for non treated sample its going to mask with the band, if there are any
So, my question is do we have to do tca precipitation for small protein sample. My size is 6.9kb with flag tag.
Can we do Bradford assay after tca precipitation because i am hoping that my quantification of protein before tca precipitation can go off after my precipitation?
I have even read somewhere that people autoclaving the membrane after transfer. I am hoping that they do it to fix the protein in the membrane. Is it done specifically for ubiquitin or can be done for any small protein?
Thanks



#174872 37kb and 7kb protein in western blot

Posted by kegaff on 14 May 2015 - 09:03 PM in SDS-PAGE and Western Blotting

Thx for your reply. I m sure its not antibody problem. I am using a flag tag. It's working perfectly with the 37kd protein. The bigger one is distinctly clear band. Comassie blue staining shows faint protein at upto 7 kb area. The front of the gel run is near that area but it seems like it is not masking the area where band should appear. Any idea what is the correct voltage of transfer for semidry transfer for such small sized protein? Is there a protocol anywhere for western for such low mw protein. I have seen people doing western with size as small as 2 kd. But they never have a detailed protocol of each n every step. Any details would be helpful.



#174868 37kb and 7kb protein in western blot

Posted by kegaff on 14 May 2015 - 02:32 PM in SDS-PAGE and Western Blotting

Hello

I have a problem detecting the small protein of 7kb in western blot. Hers is the procedure that i have been using

 

my protei has two isoforms of 37KD and 7KD with the flag tag. some other people have already show i different system and we believe it occurs in two isoforms in our system too looking at the protein sequence.

i have been using biiorad precast  tris glycine gel gradient of 4-20%. run at 180V for 45 mins. do semidry trasfer at 10V for 1 hr in 0.2micronm PVDF. i use PBS buffer and wash in PBST. the marker in the membrane are quite visible but when i do imaging, the 37kD protein only gets detected. somewhere in the forum, i saw heating the membrane in boiling water to fix the small mw protein, i tried that too but i could see nothing at 7kb band are.

What is the suggestion for doing western blot for small MW protein like 7kb?

kegaff





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