What type of proteins are you interested in isolating, i.e., glycoproteins, proteoglycans, collagens, fibronectins, entactin, nidogen, integrins, etc.? In many cases there is not a one-size fits all scenario unless you don't mind if the proteins are denatured. Then the next question would be do you just want to see how many bands there are on the gel based on general staining techniques (silver staining, PAS, luminescene) or do you want to use antibodies to selective identify the proteins? If you want to use antibodies for identification, then having the proteins denatured presents a serious problem for antibody-antigen recognition.

Also, and I don't mean to be snide, but most biochemists shy away from morphology as a method for identification. I would suggest that cryosectioning of the tissue with antibody staining would give you more usable data. Using that technology you would be able to localize selective proteins to their actual location within the tissue.

In any event, if you provide me with a little more detail I should be able to help you either way, I have a research background in both morphology  (immunocytochemistry) and biochemistry.

What type of proteins are you interested in isolating, i.e., glycoproteins, proteoglycans, collagens, fibronectins, entactin, nidogen, integrins, etc.? In many cases there is not a one-size fits all scenario unless you don't mind if the proteins are denatured. Then the next question would be do you just want to see how many bands there are on the gel based on general staining techniques (silver staining, PAS, luminescene) or do you want to use antibodies to selective identify the proteins? If you want to use antibodies for identification, then having the proteins denatured presents a serious problem for antibody-antigen recognition.

Also, and I don't mean to be snide, but most biochemists shy away from morphology as a method for identification. I would suggest that cryosectioning of the tissue with antibody staining would give you more usable data. Using that technology you would be able to localize selective proteins to their actual location within the tissue.

In any event, if you provide me with a little more detail I should be able to help you either way, I have a research background in both morphology  (immunocytochemistry) and biochemistry.

Hi Ashish, I agree with Henry in that there is no single protocol that can isolate all ECM proteins from tissues.

Many of these ECM proteins are covalently crosslinked to other ECM proteins which makes their solubilization for SDS-PAGE/western analysis next to impossible.

I think Henry has the best answer for your project, which is to stain tissue sections with Abs against specific ECM proteins.

Dear Ashish,

I agree with Steingrimur. Along those lines I attach several articles of work done in my laboratory. You will find references to the techniques you seek in those articles. 

Dear Henry and Steingrimur,

Thanks for your advice. To be more precise, I am looking for the distribution of TIMP3 (metalloprotease inhibitor) between the liver cells and ECM . According to our hypothesis, we are expecting the differential distribution of TIMP3 in our experimental setup. I was interested in preparing ECM protein lysate and cellular protein lysate and checking the protein level by Western blotting. Recently, I came across one paper which  talks about the isolation of ECM bound TIMP3 from skeletal muscles and aortas (Timp3 deficiency in insulin receptor–haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-α, The Journal of Clinical Investigation,  Volume 115, Number 12 December 2005). But, I am not sure whether the liver shares the same ECM composition like muscles.  I agree to Henry's suggestion to stain the liver sections using the antibody for TIMP3. Indeed, it will be nice if we can manage to stain the liver. 

Thanks

Hi Ashish, the paper you referred to contains this method:

"Fresh aortas were collected separately, pulverized under liquid nitrogen, and extracted with the use of ice-cold lysis buffer ...[PBS], and 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, and 0.2% sodium azide) for 1 hour at 4°C. Insoluble material was removed by centrifugation at 13,800 g at 4°C for 10 minutes. Supernatant (100 μg) was added to Laemmli buffer and heated in boiling water for 4 minutes."

They use a relatively generic RIPA buffer to extract soluble  proteins from tissues. As far as I know TIMP3 is soluble and this should work on all tissues. 

Dear Steingrimur,

If u carefully read the material and methods int he above paper,  You will find that they have used some chondroitin sulfate extractants for the precipitation of extracellular proteins. They have also used RIPA for isolation of total protein contents.

Hi Ashish, you are right. I missed that part.

The paper was following a published procedure on the purification of TIMP-3, which binds tightly to polysulfated GAGs

http://www.jbc.org/content/275/40/31226.full.pdf+html

I am a grad student trying to show the presence of Thrombospondin-1 (TSP1) on the outer membrane of extracellular vesicles (microparticles).  

TSP-1 is an ECM glycoprotein bound to proteoglycans and my PI wants me to show where it is located on the extracellular vesicles.... I am assuming when the micro particle blebs off the cell the TSP-1 blebs off with it and is bound to the membrane however am not sure how to show it. We have limited funds to play around with and so I figured cellular fractionation would be the cheapest option however cannot find a good protocol for this.  

What do you guys suggest? 

Dear Maddison Turner,

When your PI wants you to show where it is located on the extracellular vesicles, do they mean visually or biochemically? If visually, do you have access to:

1) Antibody to Thrombopondin-1?

2) Access to TEM or SEM?

3) Access to energy dispersive X-ray analysis system?

You can bind any metal with an atomic weight above 12, to the antibody. Incubate your tissue with the metal-bound antibody using similar methods as used for immunostaining for fluorescence or brightfield microscopy. Fix you tissue, section it for TEM or fractionate and coat with carbon for SEM and use the EDS system to locate the metal in specific locations in the tissue.

Alternatively, high-resolution fluorescence microscopy or high-resolution brightfield microscopy would also be able to give you a visual answer to your question as well.

Any protocol for isolation extracellular matrix proteins from decellularized liver?