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are there any good system to express functional mammalian protein? - I tried using E.coli, but protein is not functional (Aug/30/2006 )

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Thanks Scolix,
I would really appreciate if you give me an insight of your protocols, I was using Ni NTA spin columns of qiagen and amersham and got not more than unspecific binding at the end of other proteins and my protein been eluted with the flowthrough unsure.gif
I used calcium phosphate to transfect 10 cm dish and used the pellet to make the whole cell extract with different buffers and conditions (CHAPS, urea, TRIS, freeze/thawing.. name it, I did it)
How did you lyse your cells?

thanks!

QUOTE (scolix @ Sep 1 2006, 04:13 PM)
we use one 10cm plate and transfect 293 cells for expression. We have our constructs in pcDNA with the myc-Histag and purify with the Nickel column. We have been getting like 1-2 mg of protein. Actually one 10cm plate seems to b sufficient for nearly all the experiments including triplicates and controls.

let me know if u need protocol for transfection or purification.

-tertu-

Hi tertu,

I dont have it with me right now. I will send u the protocol next week.

-scolix-

Thanks a lot!!!!

QUOTE (scolix @ Sep 2 2006, 12:34 AM)
Hi tertu,

I dont have it with me right now. I will send u the protocol next week.

-tertu-

Hey Scolix!
Please make my day whenever you can

thanks

QUOTE (scolix @ Sep 2 2006, 12:34 AM)
Hi tertu,

I dont have it with me right now. I will send u the protocol next week.

-tertu-

how is your transfection efficiency?

QUOTE (scolix @ Aug 30 2006, 12:23 PM)
we usually have the His-tag in the vector so its easy to purify and yield is quite high.

-cnbeatles-

QUOTE (cnbeatles @ Sep 5 2006, 08:47 AM)
how is your transfection efficiency?


hey tertu,
I am searching for the protocol. I dont remember where I have stored it. I havent forgotten it yet and will definitely send u even if i have to retype it.

cnbeatles, our transfection efficiency is more than 90%.

-scolix-

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