Please tell the complete protocol with required centrifugation steps. I have different brain regions punched out and stored frozen at -80. Tissue sample is very less for some brain regions (around 50 mg or more).
Good morning shafa,you can follow the following procedures apart from the above mentioned protocol.
- homogenize the tissue in your homogenizing buffer (whichever you are using)-100mg/ml.
- centrifuge for 5 min.at 12000rpm at 4 degree.
- transfer the supertant into another tube.
- take bradford reading for protein conc.and prepare your protein sample accordingly from it. We are following this and we are getting good results.Hope it works for you also.
Hello Shafa,
we are using the T-PER-solution (from Thermo Scientific) for the protein extraction from frozen tissue.
Protein extraction from tissue with T-PER
- Let tissue thaw up on ice
- Weigh tissue (1g of tissue ~ 20ml of buffer)
- Homogenize tissue in appropriate amount of T-PER buffer
- Centrifuge at 10.000g for 5 minutes at 4°C
- Collect the supernatant in a new eppendorf tube.
Worked very well for us. If you have any questions, please don't hesitate to ask :).
Greetings
Hi Ajit
A similar homogenisation buffer and protocol had been used in my lab earlier. I want to study the expression profiles of some cytosolic proteins. Thanx for ur suggestion.
1- Dissolve 3 tablets of Protease inhibitor in 150ml of Ice cold PBS, use 1ml/100mg of sample
2- Homogenize with the Omni beads ruptor 24:
· Beads 19-628 (2.8mm Ceramic beads)
· Program: speed 5.65m/s
2 X 30sec. with a 15sec. pause between
3- Store overnight at -20°C.
4- Perform 2 freeze-thaw cycles to break the cell membranes.
5- Centrifuge the homogenates 5 min. / 5000rpm / 4°C
6- The supernatants were removed, assayed for their protein concentration and finally, stored at -80°C.
Please find protocol in my paper:
Hope this will help.
Let me know if u have any doubt.
Good morning shafa,you can follow the following procedures apart from the above mentioned protocol.
- homogenize the tissue in your homogenizing buffer (whichever you are using)-100mg/ml.
- centrifuge for 5 min.at 12000rpm at 4 degree.
- transfer the supertant into another tube.
- take bradford reading for protein conc.and prepare your protein sample accordingly from it. We are following this and we are getting good results.Hope it works for you also.
- Badges
- Science method