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Protein extraction from mouse organs - (Feb/08/2009 )

I want to do western blot

samples are a small piece of mouse organ (liver, kidney, lung, heart, etc)

I've done western using cell line in the past, it's really easy, just add some SDS lysis buffer to the cell pellet and boil it

But starting from tissue, I have no experience

So, basically, I am looking for a protocol to extract protein from animal organs

Concern will be like:

Are there steps before adding lysis buffer? Such as homogenization etc...
Do I use the same lysis buffer? Or change to things like NP-40...
How's the yield in general (like for each gram of tissue, use how much volume of buffer and I will get how much protein?)


Thanks a lot!!

-jiro_killua-

i just want to ask if is ot the same of tissue for animals and human????

-miechel-

miechel on Feb 9 2009, 12:38 AM said:

i just want to ask if is ot the same of tissue for animals and human????


Not sure what you meant

-jiro_killua-

Here's what I've done, using mouse tissue. Worked great.

1- Isolate organs from mouse
2- Snap freeze in liquid nitrogen (and keep at -80 if you want to process the tissues later)
3- Grind the tissues in liquid nitrogen into a powder with a mortar and a pestle
4- Add lysis buffer (the one you like, I use a slightly modified RIPA buffer) the the powder, transfer into an eppendorf and incubate with agitation for 1 hour at 4 celcius.
5- Clarifiy sample by centrifugation at 13000 RPM for 10 minutes.
6- Transfer supernatant in a new tube.

The sample is ready to use for WB (and quantification by Bradford can be done).

Hope this helps!

-madrius1-

madrius1 on Feb 9 2009, 09:27 AM said:

Here's what I've done, using mouse tissue. Worked great.

1- Isolate organs from mouse
2- Snap freeze in liquid nitrogen (and keep at -80 if you want to process the tissues later)
3- Grind the tissues in liquid nitrogen into a powder with a mortar and a pestle
4- Add lysis buffer (the one you like, I use a slightly modified RIPA buffer) the the powder, transfer into an eppendorf and incubate with agitation for 1 hour at 4 celcius.
5- Clarifiy sample by centrifugation at 13000 RPM for 10 minutes.
6- Transfer supernatant in a new tube.

The sample is ready to use for WB (and quantification by Bradford can be done).

Hope this helps!


That sounds very promising!!
thanks a lot

Just a practical question: What's the yield? (How much lysis buffer add to how many mg of tissue and got what concentration of protein?)

And how to grind the tissue with liquid nitrogen?
Like sit the mortar on some LiqNi? Is that feasible (would it break if the temp is too low, etc)

-jiro_killua-

Someone told me about PIERCE's T-PER Tissue Protein Extraction Reagent, and the manual of it says:

use 20ml of buffer per 1gram of tissue, and homogenize the tissue in the buffer

How does that sound?

-jiro_killua-

No, don't put the mortar in liquid nitrogen. It would probably break.

What I did is to put my frozen samples in the mortar, and then add liquid nitrogen. You have to grind the tissue while the liquid nitrogen is in the mortar. Re-add liquid nitrogen when needed.



As for the amount of lysis buffer, I couldn't tell you any exact measures. I added sufficient volume of buffer to recover most of the powder, but not too much, as I didn't want my sample to be too diluted.

I ended up with 1-2ml of sample, at around 2-3 g/l.

One step reagents could be another way, yes. I would worry about the recovery though.

-madrius1-