Muscle Homogenate

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Guy Sovak
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Muscle Homogenate

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Hi All,
Starting to work with muscle tissue, Psoas muscle from pork.
I would like to run homogenates from the muscle and connective tissue portions.
Any one knows how to do perform muscle homogenization and did it before.
There are few articles with different methods. Either smashing the sample after it is frozen in liquid nitrogen or homogenize it whole fresh.
Any insights, ideas, protocols?
Thank you all
Guy

Kvachhani
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Hi, i have done tissue

Hi, i have done tissue homogination (for liver and spleen), for that this liquid nitrogen mixing and smashing in  mortar and pestle. you have to add some part of tissue with adding small quantity of LN, add LN as per requirement. This works well.
Initially did it with a small quantity.i have another protocol but no experience on this.
http://www.sacmm.org/pdf/Animal%20Tissue%20Homogenization.pdf
www.protocol-online.org/biology-forums/homogenization.html
regards

Arvind Singh Pundir
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guy wrote:

guy wrote:

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Hi All,
Starting to work with muscle tissue, Psoas muscle from pork.
I would like to run homogenates from the muscle and connective tissue portions.
Any one knows how to do the homogenization and did it before.
There are few articles with different methods. Either smashing the sample after it is frozen in liquid nitrogen or homogenize it whole fresh.
Any insights, ideas, protocols?
Thank you all
Guy

 
Hi Sir
please go to the following links prbably they may help
http://www.sacmm.org/pdf/Animal%20Tissue%20Homogenization.pdf
http://www.freepatentsonline.com/7270284.html
http://www.biochemj.org/bj/077/0326/0770326.pdf
if you can access the following paper
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45BC6SH-6B&_user=1633819&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000043301&_version=1&_urlVersion=0&_userid=1633819&md5=7b32a0c0592b7e74340a3d42a56238f2
hope they aid to your help
arvind

Guy Sovak
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Dear Pundir and Kavacchani,

Dear Pundir and Kavacchani,
None of the protocols that you have given there link are helpful. Not dealing with Muscle.
One of them even speaks about tissue with Aspergilus contamination
Please look at the protocols that you are sending before sending them.
I realy apriciate your help and still loking for a good Muscle homoginization protocol.
Thanks
Guy

R Bishop
R Bishop's picture
What do you plan to do with

What do you plan to do with the tissue?  Gene expression? protein isolation? Glycan analysis?  The method employed to homogenize the muscle and connective tissue hinges upon the details of the experimental work flow you need to follow.
 

Guy Sovak
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I want to start with a very

I want to start with a very simple question regarding quantification of different  proteins  in the muscle and connective tissue, at couple of cross sectional planes of the Psoas Major Muscle.
Some preliminar bimechanical studies shows difference in axial movement of the muscle, one idea is that the amount of proteins like fibronectins, Collagen's are not the same at different cross sectional planes.
So I would like to start with a total homopgenate of muscle (muscle and connective tissues) and then go specificly to either ECM or cellular portions of both tissues.
 
Guy

R Bishop
R Bishop's picture
Sorry for the delayed

Sorry for the delayed response Guy.
In that case, I would go with a basher technique first like solubilizing the muscle tissue in a homogenizer and immediately adding in 4M guanidinium chloride overnight at 4C.  This will break up all protein-protein and protein-lipid interactions.  You then do a low speed spin to remove the nuclei etc. and dialyze the guanidinium out.  This in my hands the best way to examine extracellular matrix proteins like fibornectin etc.  If you are interested, I will paste the protocol in here when I get back to the lab tomorrow. Let me know.
 
Rus

Guy Sovak
Guy Sovak's picture
Thanks Rusty,

Thanks Rusty,
Yes please post the protocol.
Please let me know what type of homogenizer do you use.
Also please post the dialysis procedre are you using a dialysis chamber?
Thanks
Guy

R Bishop
R Bishop's picture
Guy,

Guy,
Here's my protocol for estracellular matrix protein extraction from muscle tissue. Sorry it took me so long to dig it up.
 
1. Mince the muscle tissue with a razor blade as best as you can and immediately add 50 mM NaAc, pH 6, 0.1 M NaCl, 0.3% CHAPS, protease inhibitors to appropriate volume (10mls/gm) and grind the tissue in homogenizer (Polytron).  It will be a little sticky so give it a few pulses and return to ice in between.
2. Extract in 4 M guanidine HCl, 50 mM NaAc, pH 6, 0.1 M NaCl, 0.3% CHAPS, protease inhibitors for 24-48 hrs @ 4C
3. Dialyze into 6 M urea, 50 mM NaAc, pH 6, 0.1 M NaCl, 0.3% CHAPS @ 4C with 3 changes of buffer (50 ml : 2000 ml   {1:40 dilution 3x ‡  brings to 60 mM GuHCl}
4. Clear debris by centrifuging in a tabletop centrifuge, 10 min at 4000 rpm
5. Filter extract through Whatman No. 1 filter paper
From here you will have to purify proteins based off chromatography or antibody pull down or western detection ec.
 
Rus
 

Ivan Delgado
Ivan Delgado's picture
Hi Guy,

Hi Guy,
In the past I used to work with muscle samples from mice (possibly similar but maybe not). My main interest was to extract high quality RNA for expression analysis, so it was very important for me to treat my samples as delicately as possible, which is why I did not want to freeze and thaw my samples. The method that worked for me was the use of a hand-held glass dounce homogenizer. It requires you to push and pull quite a bit, but it also gives you a lot of control over how much to homogenize and since it is glass you can easily see the whole process, which can be critical for tissues like muscle that may not homogenize very easily. Here is a link to a good example: 
http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=9578358

Guy Sovak
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Thanks Ivan,
and Thanks Rusty,
I think that I will try the protocol that Rusty used as it would be more apropriate for me.
Rusty ,
In the dialysis section how do you do it? Using a cassette or eppendorf dialisis tubes (what is the MW cut of ) or maybe other means?
We shell se, I will order the chemicals and then I report what were my results.
Guy

MegCD
MegCD's picture
Thanks for the post Ivan!  I

Thanks for the post Ivan!  I was looking for a good protocol for muscle homogenization to use with RNA applications. I've been trying to get good yields from small bits of mouse muscle, but have thus far been unalbe to get decent yields.  I'll try using the glass homogenizer.
Thanks,
Meg

mouri
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hi,

hi,
i am sorry if this is the wrong thread for this qs, but i have a problem with smooth muscle homogenisation. see, i am working with antioxidant enzymes and oxidative stress related damage. can anyone please provide a pointer for me for which protocol i should use for this purpose?
 
thanks in advance.
mouri