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Western blot secreted protein control

Western blot secreted protein control - Western Blot Forum

Western blot secreted protein control - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 01-21-2009, 03:06 PM
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Default Western blot secreted protein control



Hi,
I am currently performing western blots on my protein of interest, which is about 300kDa in size. The protein is secreted from my cell line and so I collect the cell media, concentrate my protein by spinning down the media in filter centrifuge tubes, and then perform the western on the concentrated material.
I have been able to detect my protein this way, however now I am attempting to suppress production of the protein. In order to show this suppression (ie the difference between samples treated with targeting and non-targeting constructs) I need a loading control, but I am having difficulty finding something suitable. Preferably I need a high molecular weight protein which is secreted from my cell line, but I have not been able to find something suitable in the literature.

I was thinking of adding a certain amount of a known protein to each of my concentrated media samples (ie targeting and non-targeting) and then probing for this protein with an antibody during the western. This should tell me if I get equal loading in all wells of my western gel.
If the media I have used was also taken from cells which were 100% confluent in all cases, then should all this be enough of a control to prove that differences between my targeting and non-targeting developed bands are due to suppression?

Also does anyone have any suggestions of what protein standard I would be able to add to my samples and probe for? BSA would be ideal as I have some available here in my lab, but I think it is too small as it would probably be run off my gel, and so I need something larger.

I'd really appreciate opinions/advice.
Thanks
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  #2  
Old 02-11-2009, 03:01 PM
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Default Re: Western blot secreted protein control

Would a picture of a PonceauS stained membrabe or Coomassie stained gel which show equal protein levels be enough or do you have to have a specific protein?

Last edited by Liana; 02-11-2009 at 03:03 PM. Reason: typo
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  #3  
Old 12-02-2010, 03:57 PM
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Smile Re: Western blot secreted protein control

Quote:
Originally Posted by alphabetty View Post
Hi,
I am currently performing western blots on my protein of interest, which is about 300kDa in size. The protein is secreted from my cell line and so I collect the cell media, concentrate my protein by spinning down the media in filter centrifuge tubes, and then perform the western on the concentrated material.
I have been able to detect my protein this way, however now I am attempting to suppress production of the protein. In order to show this suppression (ie the difference between samples treated with targeting and non-targeting constructs) I need a loading control, but I am having difficulty finding something suitable. Preferably I need a high molecular weight protein which is secreted from my cell line, but I have not been able to find something suitable in the literature.

I was thinking of adding a certain amount of a known protein to each of my concentrated media samples (ie targeting and non-targeting) and then probing for this protein with an antibody during the western. This should tell me if I get equal loading in all wells of my western gel.
If the media I have used was also taken from cells which were 100% confluent in all cases, then should all this be enough of a control to prove that differences between my targeting and non-targeting developed bands are due to suppression?

Also does anyone have any suggestions of what protein standard I would be able to add to my samples and probe for? BSA would be ideal as I have some available here in my lab, but I think it is too small as it would probably be run off my gel, and so I need something larger.

I'd really appreciate opinions/advice.
Thanks




Hello,

I am currently doing an experiment of the same type,my protein is secreted from my cell line and so I collect the cell media, concentrate my protein by spinning down the media in filter centrifuge tubes, but i can not quantificate it in a spectrophotometer, because my concentrate have color due to the media, what can i do for eliminate the color of my sample and then do western on the concentrated material.


Thank you
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  #4  
Old 02-08-2011, 03:08 PM
Pipette Filler
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Default Re: Western blot secreted protein control

Try heatshock protein, HSP90, GAPDH, or beta-actin.
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  #5  
Old 02-20-2011, 01:33 PM
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Default Re: Western blot secreted protein control

heat shock, and beta actin are good controls, altough indirect

(you can show from the cell lysate from the cells - then you proove that you have the same amount of cells.)

but from the supernatant it may be difficult to find one (secreted)
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