Western blot secreted protein control

[alphabetty]

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

[Liana]

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?

[Natei]

Originally Posted by alphabetty

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

[Cancer Biologist]

Try heatshock protein, HSP90, GAPDH, or beta-actin.

[gehrs]

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)