would depend on what tissue you want to analyse and what sort of proteins u want to look for. Mosst easy way is to cut out the tissue, wash in PBS and put in liquid nitrogen, After N evaporate you can grind the tissue in a common lysis buffer (with usual ~1% triton and protease inhibitors) with pestle/mortar or a mechanical device. Spin down the pellet and use the supernatant. YOu should do bradford -type assay to quantify total proteins This works for most of the proteins and most of the tissues.

Hi, for me using the rat aorta tissues, similar procedures conducted by using RIPA lysis buffer and using pestle to homogenize it. I found my tissues sample wouldn't be homogenized completely and resulting low protein yield, i would like to ask any suggestion for this kind tissues?

Are u using Liq N to flash freeze the sample. After this the soft tissues become so brittle that they will break like glass in the homogenizer. There lll be still a lot of debris, but usually it should work. In our lab we do this for most of the tissues(lung, liver, spleen, brain, muscle, intestine, heart etc. -for muscle we use an electrical homogenizer). But this way I had problems in getting keratins from intestinal tissue. Since it is a very soft tissue, we then minced it and boiled in 2x SDS loading buffer and used the supernatant- but sometimes gels run weared with these lysates.

I tried with a few samples, flash freeze in liquid nitrogen and thawed in 37 C water bath. As you said, there is still a lot debris, and the protein yield is low for me. for the soft tissues like spleen, how long do you need to take for homogenized it?

In your procedure you are relying completely on freeze-thaw based lysing, but you ll still have problem in grinding the slimy tissue. What I do is to PBS wash the tissue and the dry tissue is kept on a mortar. directly some liq N is added and aallowed to evaporate. The tissue now will be like a brick (white and sometimes sticks to the mortar). You can at this stage break it with pestle (slowly otherwise it flies out!!!). It will get crushed like rock. Now keep the pestle on ice and add ice cold lysis buffer (we use a lysis buffer with 1% triton- I think RIPA should work, but if you want to try, you can try triton- I can send you my recipe too). Now grind it ..the time required is variable. But liver and spleen could be very easily crushed this way. As I mentioned before we get a lot of debris (could be extra cellular stuff too) so the yield is not uniform but extraction is really good.

I jut had a look. SIGMA and PIERCE sell RIPA buffer and in their data sheets they mention only about cultured cell lysis, no tissue. So may be NP40-SDS mixture in RIPA is too mild.(thats y good for IP-pulldowns etc etc)..I have no experience with it, but may be this is the reason for low yield..

OK, I ws working a bit on spleenocytes and I am using a slightly different procedure that yeild good amount of protein.

as you said I flash freeze the spleen after dissection. use a homogenizer sieve with pistol in ice cold Pbs after a couple of washes with ice cold Pbs keeping the pellet I count the cells in sol. I usually divide the samples into 5-10 million cells per aliquots and then add RIPA modified from a recipe I got from Cold spring harbor website, PMSF as a proteinase inhibitor is added fresh.

I leave it on ICE (4 degrees) for 30 min .... put it on a vigorous shaker for another 30 min.

This vigorous shaker is not like the ordinary shaker its some shaker that we made out of vortex machine and we removed the top and added some home made tray to hold the epps. firmly during the vigorous shaking. after that spin in centri. for 15 min to get the splashed fluid. finally you could see tissue debris pellet that may disturb you, a college of mine grind these pellets and re centrifuge, but I usually dont do that and the protein yield is OK.

hope its helpful

Thanks for u advice. I used RIPA lysis buffer 1X sc24948 from santa cruz. I will try as u suggested in my next extraction. I also came across the method which add in N inside the tissue, but they mentioned have to be in dry ice, so i never try it. It would be great if u can lend me ur lysis buffer ingredients as a backup next time

this is the one I am using:

http://cshprotocols.cshlp.org/cgi/content/full/2006/24/pdb.rec10617?maxtoshow=&hits=10&RESULTFORMAT=&fulltext=RIPA&searchid=1&FIRSTINDEX=0&resourcetype=HWCIT&text_only=true

it does not contain Na Azide as your recipe

Hi, great, I am excited to try out the protocol from both you. Hopefully the luck is with me this time. Thanks

Our normal buffer composition is

20 mM Tris pH 7,0: 0,1 mM EDTA :1 mM EGTA: 1 mM Na-o-Vanadat: 10 mM beta-Glycerophosphat: 50 mM NaF: 1% TX-100

0,1% beta-Mercapto / 0,1 mM DTT (Better to add fresh (we some times make complete and freeze at -20). Add 1 protease inhibitor pill (Roche)/10mL before use

( I think most of the components are to maintain the phospho-proteins properly). A normal std buffer like PLC should do.

Good luck

Dear Gayatri

There are several methods for protein extraction from the cells and tissues for protein studies such as WB:

1. Grinding/hemogenization (such as mortar and pestle, blender, tissue hemogenizer)

2. French press

3. Enzymatic lysis

4. Sonication

5. Osmotic shock

6. Freeze-thaw

7. Detergents

Dear Rahul

By using each methods, proteins must be soluble before SDS-PAGE.

To prevent protein lysis by protease you must use protease inhibitors or work with liquid nitrogen.

If you need the standard protocol for WB, please dont hesitate to contact me.

The answer depends on what you are planning to detect. The buffer will be different depending on whether you are looking for a soluble protein or a membrane protein. For soluble proteins, a salt buffer and a homogenizer will work fine. For membrane proteins you will need detergent. Again the amount of detergent required and the type of detergent will vary with the protein of interest. A good start would a general protocol would be

10mM HEPES/Na pH 7.4

10% glycerol

1% Triton X-100 (or Nonidet-P40)

SDS should generally be avoided in cell lysis buffers as it easily breaks open nuclei and all you are left with is a useless sticky gunk

http://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1