Hello Jyoti,

As soon as you have isolated your tissue, you should either immideatly freeze your sample in liq.N2 for storage, or chop it in small, 1mm pieces and then crosslink it in PBS containing 1% formaldehyde.

Personally, i would quickly rinse the intestine with PBS to remove any residues of intestinal contents, then snap freeze the remaining tissue and grind it in N2 with a mortar&pestle. You then collect the powder in a falcon already containing ~10ml PBS 1x with 1% formaldehyde and let it crosslink 10 - 15min on a shaker at RT. Quench it in glycine for 5 min, spin down saple and wash 1-2 times with PBS before continuing with further lysis steps.

If it's feasible for you, try adding protease inhibitors already to the PBS and use it ice cold to prevent protein degradation.

This procedure worked for different kinds of tissues for me so far.

good luck!

I agree with alex, In my view, take fresh tissue rinse with 20 pbs several times ( 5-6 times) and chop it into small pieces and cross link with 2% formaldehyde (methanol free) for at least 20 min and add glycine. it is easy to use plastic pestle and hand held motor to homogenize in chip buffer (500 ul) containing protease inhibitor cocktail (1x). incubate sample at room for few mint and centrifuge to remove soluble proteins. The pellet should be redissolved in chip buffer containing protease inhibitor cocktail and sonicate for 30 sec pulse with 20sec rest for 20 to 30 min. After sonication, take 2 to 3 ul and run agarose gel to check appropriate shearing. You can modify pulse time based on shearing results....once you confirm shearing is standardize, use magnetic beads for down stream experimen.

RKG

Thanks, Alex and Rajendra. I will try both and see which one works for me. Thanks again..