Hello, I also think your problem is the dissection, not the lysis. Intestine is one of the dirtiest tissues, the small intestine receives the pancreatic juice, made to enzymatically destroy proteins. We proceed similarly to Thomas, but we place the duodenum in iced PBS (we freeze 1x PBS at -20°C and then let it thaw and shake it only as much as we get a dense icy liquid... this is far below 0°C!). Then we flush it with the same iced PBS, gently, not to disrupt the mucosa, but thoroughly. Finally we open it longitudinally and wash it in the iced PBS.

After this you can briefly dry it by touching a piece of adsorbent paper and either freeze it in liquid N2 and store it at -80°C for future use, or directly place it in 2x Laemmli buffer (volume will depend on the size of your tissue, we lyse the duodenum - 1.5 to 2 cm long - in 5-600 ul), homogenize it with a teflon douncer and boil it for 10 min vortexing for 15-20 seconds every 2 min to fragment the DNA. Cool on ice and spin 5 minutes at max speed to remove whatever did not solubilize, which will be very little!

In this way you have all possible proteins, from membrane to nuclear, for western blotting.If you will plan experiments where you need the native proteins (IPs, pull-downs, etc.) you'll have to change extraction method, but this gives you the best quality proteins. Keep in mind that some proteins do not tolerate being boiled. If your proteins of interest are of this type, you can sonicate your extract after douncing, as already suggested by Thomas, and then spin or ultraspin.

You can quantify the proteins in 2x Laemmli buffer diluting 1:10 the protein lysate and reading 10 ul of the dilution (which corresponds, in fact, to reading 1 ul of the lysate... but the trick dilutes the SDS to below 0.2% so that it does not interfere with the Coomassie in your Bradford. Don't forget to do the same with the blank).

Good luck.

sonicate the sample during protein extraction. 

I want to know your enzyme digestion cocktail recipe. 

Do you get any bands from house-keeping genes like beta-actin or GAPDH? 

Rinse out the intestine with saline (for mice we use a 1 mL pipette tip) with protease inhibitors. On ice, fillet open the intestine on a glass plate on ice. Scrape the luminal surface (mucosa) with a glass slide (mild pressure). You should get a wet sticky little glob that you can save in a polypropylene tube --flash freeze in liquid nitrogen and store in -80 freezer until ready to analyze. The remaining tissue can be discarded. You might want to save the luminal rinse to analyze microbiota. We then homogenize the sample with Teflon on glass in a protein lysate containing protease inhibitors, sucrose, EDTA and DTT. I then sonicate the samples. I also like to ultracentrifuge at 30,000 rpm for 1 hour, and remove the lipid and resuspend in the same lysate (assuming it is not a lipid droplet protein). I then run proteins assay (did you get protein?), dilute all samples to equal concentrations, add Laemmle containing reducing agent (beta-mercaptoethanol), before adding to gel. There are other ways to lose proteins. Let me know if this helps.

Hello, I also think your problem is the dissection, not the lysis. Intestine is one of the dirtiest tissues, the small intestine receives the pancreatic juice, made to enzymatically destroy proteins. We proceed similarly to Thomas, but we place the duodenum in iced PBS (we freeze 1x PBS at -20°C and then let it thaw and shake it only as much as we get a dense icy liquid... this is far below 0°C!). Then we flush it with the same iced PBS, gently, not to disrupt the mucosa, but thoroughly. Finally we open it longitudinally and wash it in the iced PBS.

After this you can briefly dry it by touching a piece of adsorbent paper and either freeze it in liquid N2 and store it at -80°C for future use, or directly place it in 2x Laemmli buffer (volume will depend on the size of your tissue, we lyse the duodenum - 1.5 to 2 cm long - in 5-600 ul), homogenize it with a teflon douncer and boil it for 10 min vortexing for 15-20 seconds every 2 min to fragment the DNA. Cool on ice and spin 5 minutes at max speed to remove whatever did not solubilize, which will be very little!

In this way you have all possible proteins, from membrane to nuclear, for western blotting.If you will plan experiments where you need the native proteins (IPs, pull-downs, etc.) you'll have to change extraction method, but this gives you the best quality proteins. Keep in mind that some proteins do not tolerate being boiled. If your proteins of interest are of this type, you can sonicate your extract after douncing, as already suggested by Thomas, and then spin or ultraspin.

You can quantify the proteins in 2x Laemmli buffer diluting 1:10 the protein lysate and reading 10 ul of the dilution (which corresponds, in fact, to reading 1 ul of the lysate... but the trick dilutes the SDS to below 0.2% so that it does not interfere with the Coomassie in your Bradford. Don't forget to do the same with the blank).

Good luck.

I would also suggest acetone precipitation of the proteins prior to adding loading solution - to remove bile acids and other lipids that may cause problems during electrophoresis.

Thanks all answers. Sorry for delay.

Dear Alex Chung Cheung, we use normal RIPA buffer for lysis. But only we add protein phosphatase inhibitor. We just got weak ß-actin bands in our previous experiment.

I examine which your helps and search on internet differences. I'll share our results.

Thanks again for all answers. Have a good experiment!

Ediz.

Dear Ediz Coşkun,

have you succeeded with finding the best homogenization procedure? I would also be interested...

Best and thanks,

Annekatrin

Dear Annekatrin,

Although there weren't sufficient intestine samples for doing homogenization we couldn't try homogenization ways shared above. We obtained homogenizated small intestine samples from research group that we work together. So, I didn't ask them how to do that, because we got bands in western blotting. I can ask them if you want how to do that in detail, it seems like that you have a trouble like us.

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