Sulail Fatima

After making the 1x lysis buffer take 100mg of grinded tissue and add 930uL of lysis buffer 1mM of PIC 10uL, 10mM OF PMSF 10uL and 50uL sodium deoxycholate. Hence, the total volume is 1mL, and the sample is homogenised by a sonicator or mechanical pestle and ice bucket. Centrifuge 16000 rpm for 20min at 4 C . Collect the supernatant and check the protein concentration.

Good Luck!

Dear Dr. Sulail Fatima

To remove fat while extracting proteins from tissues, the most common method is to use a cold acetone precipitation step where you add a large volume of ice-cold acetone to your tissue homogenate, which will precipitate the proteins while leaving the lipids in solution, allowing you to remove them by centrifugation. You can then resuspend your protein pellet in your desired buffer for further analysis.

Some useful tips that may help.

a. Ensure that you add large volume of acetone (5-10 times the volume of your sample) to maximize lipid removal.

b. After adding acetone, vortex thoroughly and centrifuge at high speed to pellet the proteins.

c. You should remove visible fat from the tissue sample before homogenization.

d. You should use a lysis buffer containing a detergent that is less likely to solubilize lipids such as Triton X-100, in appropriate concentrations.

e. Perform multiple centrifugation steps at different speeds to separate the protein-containing supernatant from lipid-rich phases.

I have attached a protocol below which you may want to follow.

Part I: Homogenization

Part II: Tissue lysis and centrifugation

1. Add Triton X-100 to a final concentration of 1% (v/v), mix well, and keep on ice.

2. Incubate at 4°C for 30-60 min.

3. Centrifuge at 12,000 × g for 15 min at 4°C.

4. Remove upper layer of lipid and transfer the supernatant to a new 1.5-ml tube.

5. Repeat Steps 3 and 4 twice to get rid of maximum amount of lipid.

6. Store the extracted sample at -80 °C until you carry out protein estimation (BCA assay).

7. Perform Western Blotting.

Regards,

Malcolm Nobre

You should do it in ice.

The choice of temperature for protein extraction depends on the properties of your sample and the downstream application. For tissues with high fat content and to ensure effective solubilization of both proteins and lipids for western blotting, using a boiling hot (95-100°C) temperature is optimal. The SDS in your lysis buffer is highly effective at denaturing proteins and solubilizing lipids at high temperatures, which helps overcome the interference caused by fat in BCA assays and ensures consistent protein extraction. While room temperature (RT) or ice-cold conditions can preserve protein functionality for enzymatic assays or sensitive applications, these are less efficient at solubilizing lipids and may lead to variability in protein yield and loading. Therefore, heating the samples with your SDS-based buffer will likely yield the maximum protein for your application, especially since you are denaturing the proteins for SDS-PAGE and western blotting.

I have always done extractions cold or near room temperature. With a 1% SDS (which is very high), you would have no trouble extracting, at least for mammalian cells in either whole tissues or culture. The big problem with using SDS or saponan (or other ionic detergent) greater the .5% is the complete dissolution of the nuclear membrane which then makes you crude extract very viscous (due to the released DNA) and difficult to work with (i.e. centrifuging the lysate to get a clear supernatant).

Thanks everyone for the inputs. Fat problem is resolved but I am facing some trouble while pelleting tissue debris. I am using the attached protocol. After centrifugation and removing supernatant, I store my supernatant in -80c until bca estimation which is usually taking place in a day or two. I have observed that when I thaw my supernatant on ice and centrifuge again before bca, I get a pellet (again). This time it is little bigger than the pellet which I got after my first centrifugation. Is the speed, time of centrifugation or some other factor contributing to this observation.

in addition, bca looks fine almost the same protein I used to get with other protocols I did. Moreover, I ran them on gel and did housekeeping protein b actin which also looks fine. But I am just worried why the pellet keeps coming everytime I would centrifuge even though I clearly and very precisely separate the supernatant first time when I am performing extraction of protein.