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37kb and 7kb protein in western blot


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#1 kegaff

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Posted 14 May 2015 - 02:32 PM

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



#2 bob1

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Posted 14 May 2015 - 08:10 PM

I take it you have stained your gel to ensure that the 7 won't have run off the gel?

 

7 kDa is quite small, you could try layering 2 membranes in your transfer in the hope that the second will retain when it passes through the first. You could also try playing with the transfer time to see if 7 kDa is retained on the membrane or if it is passing through.

 

Are you sure this isn't an antibody problem - try a dot-blot to see if you can get a positive signal with the antibody.



#3 kegaff

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Posted 14 May 2015 - 09:03 PM

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.

#4 bob1

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Posted 15 May 2015 - 12:06 AM

There isn't any special protocol, it is a matter of playing with the conditions yourself, as the conditions can vary according to the protein. In general, the time and voltage/amperage are the ones to play with for transfer problems. You sould also ensure that you have 20% methanol in your transfer and you could try leaving out the SDS.



#5 mdfenko

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Posted 15 May 2015 - 03:32 AM

those who transfer a 2 kDa peptide probably us tris-tricine sds-page to separate. i would recommend that you look into trying it, i've had a lot of success using it for my peptides.

 

transfer time for very low molecular weight proteins is short. check the manual that came with your transfer apparatus, it should give you all the information you need to successfully transfer your protein. my transfers were for 15 minutes or less (again, depends on the apparatus).

 

you may want to try a gradient tricine gel so that transfer will be even for both 37 and 7 kDa proteins.


Edited by mdfenko, 15 May 2015 - 03:35 AM.

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#6 kegaff

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Posted 22 May 2015 - 09:37 AM

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

#7 kegaff

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Posted 23 May 2015 - 07:29 AM

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.

#8 mdfenko

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Posted 25 May 2015 - 08:38 AM

in what buffer do you isolate the protein? the components of that buffer may be interfering with the migration pattern of the lower molecular weight proteins.

 

also, when you precipitate with tca, you should ensure that the sample is properly neutralized.


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#9 kegaff

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Posted 25 May 2015 - 12:56 PM

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.

#10 kegaff

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Posted 25 May 2015 - 01:00 PM

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?

#11 mdfenko

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Posted 26 May 2015 - 04:13 AM

the amount of protein you're loading should be fine.

 

detergents disrupt migration of proteins, especially smaller size proteins. also, triton will displace sds.

 

you should try to completely solubilize the pellet. the smaller sized proteins don't necessarily preferentially solubilize.

 

one other thing, many protease inhibitors are ploypeptides. some of them may be migrating along with your 7 kDa protein.


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#12 kegaff

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Posted 26 May 2015 - 10:07 PM

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?

#13 mdfenko

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Posted 27 May 2015 - 03:38 AM

you can continue to use the lysis buffer but then you should either precipitate the protein and replace the buffer with one that won't interfere with electrophoresis or dialyze to remove the problematic components.

 

years ago we worked with a protein which was only soluble in a high salt environment. in order to apply the protein to sds-page we would dialyze the protein against sds sample buffer (without tracking dye and glycerol which were added later). this way we removed the salt while we denatured the protein. you can do something similar to remove the detergents, just make sure the dialysis membrane won't pass the proteins of interest.


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#14 kegaff

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Posted 27 May 2015 - 05:09 AM

Thanks for your advice. I ll try that way.

#15 kegaff

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Posted 27 May 2015 - 09:56 AM

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.




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