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western blotting loading control


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5 replies to this topic

#1 cn981

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Posted 21 June 2009 - 08:00 AM

i want to quantify the target protein locats in the membrane.can i choose the actin or GAPDH as the internal protein for the laoding control?are there actin or GAPDH in the membrane?please share your advices with me.thank you.

#2 bob1

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Posted 21 June 2009 - 05:22 PM

If you are doing whole cell lysis GAPDH or actin will be fine. If you are doing clean membrane preparations then neither will be fine.

#3 cn981

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Posted 21 June 2009 - 06:37 PM

If you are doing whole cell lysis GAPDH or actin will be fine. If you are doing clean membrane preparations then neither will be fine.

I am purifying the cell membrane.It is clean membrane preparations in theory.So what should I choose as the internal protein for the loading control.Some friends adivised me the Galpha for the loading control.Is it ok?What else I can get?Thank you.

#4 victor.m

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Posted 22 June 2009 - 12:38 AM

Hi cn981,
I do not know what is the best loading control for WB of total cell membrane proteins. My experience, for example, I had a protein anchored in plasma membrane, so I isolated crude plasma membrane and as loading control I used Na/K ATPase a typical plasma membrane protein. May be, it is not typical loading control for plasma membrane (or total cell membrane generally) but it seemed to work in my experiments.
GAPDH and actin are not membrane proteins (GAPDH is cytosolic marker). At the web site of Abcam.com you could find helpful information.

vic

#5 cn981

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Posted 22 June 2009 - 07:49 AM

Yes,you are right.The Na/K ATPase is used for the loading control when quantify the membrane protein.I have another question that what is the difference between the membrane proteins and the particulate fraction proteins.Some articles studied the protein kinase c pointed out this protein translocated from Soluble Components to particulate fraction.

#6 cn981

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Posted 23 June 2009 - 04:50 AM

Will someone give me suggestions?Thank you.




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