The basic protocol is very easy:
- homogenate (I use UltraTurrax homogenator) the tissue in RIPA buffer (recipe everywhere in the internet)
- leave the homogenate 30 min on ice
- if you want you can sonicate the lysate to shear the genomic DNA (it will interfear with the gel running)
- centrifuge 10 min, full speed, in the cold room (or in cooled centrifuge)
- take the supernatant and check the protein concentration (if necessary)
- dilute the supernatant with Laemmli buffer (recipe everywhere in the internet) and incubate 5 min at 70 C to denaturate all the proteins (including proteases)
- you have your sample ready for western blot
NOTE 1: keep the samples on ice all the time. To avoid activity from the endogenous proteases.
NOTE 2: it is very important to add proteases inhibitors to the RIPA buffer right before homogenizing the tissue, otherwhise you will have all your proteins digested by endogenous proteases. Many vendors (Roche, Biorad...) cell proteases inhibitor tablets to be dissolved in water and then in the RIPA buffer. Besides the inhibitors I can suggest to add also DTT (final conc 1 mM) and PMSF (final conc 0.2 mM) to the RIPA buffer to complement the other inhibitors.
NOTE 3: chemical inhibitors (DTT, PMSF) have a limited time action, they are active for 1-1.5 hours and then you have to add more.
NOTE4: after you mix the lysated with Laemmli buffer your all the proteins are denaturated and quite safe.
Thank you for your suggestion but sir ..I need the protocol for isolated murine liver tissue lysate..meas the tissue sample preparation for western blot......
The basic protocol is very easy:
- homogenate (I use UltraTurrax homogenator) the tissue in RIPA buffer (recipe everywhere in the internet)
- leave the homogenate 30 min on ice
- if you want you can sonicate the lysate to shear the genomic DNA (it will interfear with the gel running)
- centrifuge 10 min, full speed, in the cold room (or in cooled centrifuge)
- take the supernatant and check the protein concentration (if necessary)
- dilute the supernatant with Laemmli buffer (recipe everywhere in the internet) and incubate 5 min at 70 C to denaturate all the proteins (including proteases)
- you have your sample ready for western blot
NOTE 1: keep the samples on ice all the time. To avoid activity from the endogenous proteases.
NOTE 2: it is very important to add proteases inhibitors to the RIPA buffer right before homogenizing the tissue, otherwhise you will have all your proteins digested by endogenous proteases. Many vendors (Roche, Biorad...) cell proteases inhibitor tablets to be dissolved in water and then in the RIPA buffer. Besides the inhibitors I can suggest to add also DTT (final conc 1 mM) and PMSF (final conc 0.2 mM) to the RIPA buffer to complement the other inhibitors.
NOTE 3: chemical inhibitors (DTT, PMSF) have a limited time action, they are active for 1-1.5 hours and then you have to add more.
NOTE4: after you mix the lysated with Laemmli buffer your all the proteins are denaturated and quite safe.
Thank you...Matteo Da Ros
I will try..from tomorrow..
I was tried ....one cell lysate buffer nuck buster from merck...but I dont see any band in wb...thank you once again
Perhaps your protein is not expressed in liver or your antibody is not working.
Hello Subhadip Das,
i achieved very good results with the thermo "NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit" in the nuclear fraction. Maybe you should also try to locate your protein and isolate the specific fractions where your protein is located/enriched.
good luck :)
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