I tried sonication and homogenizer and then pestle and mortar method as well but the protein concentration is very low bcz of which I am unable to get bands.
Are you also using a lysis buffer such as RIPA? We usually get good results when using an ultraturax in Ripa.
You could also try cooling a motar and pestle in liquid nitrogen as well as the tissue und then crush the tissue while keeping it under nitrogen, tranfer to RIPA and proceed as normal.
I found it to work quite well with stubborn tissue.
I am using RIPA with PIC. Thank you Gensch for your valuable suggestion. I will try with this.
I am used T-PER tissue protein extraction solution from thermo scientific, first tissue samples were homogenated with biospec homogenizer for 3-5 times at maximun speed (human adipose tissue, for 100 mg tissue 500uL of solution),under 4 degree condition, then spun at 8000 rpm for 15 min, sup were separated into a fresh vial then 25 uL of 100mg/mL protease inhibitor (Roche) added into the 450-500 uL of sup and sonicated for 2 cycle at lower frequency.
I got very satisfactory results from the above protocol, if possible try.
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