Hi Researchers,
Currently I am doing 2DGE to identify differential protein expression (unknown) in plant tissues under various abiotic stress conditions. I am following phenol extraction method for protein extraction. After dissolving the final pellet in 2D lysis buffer i am getting sample in yellow color which means the pellet itself is yellow in color. I want to know whether it will affect isoelectric focusing of the protein or i can use the sample as such or i should purify the sample. If it is so kindly suggest me good purification protocols for plant protein. Thanks in advance.
Hi
To extract proteins from plants I use following conditions:
Plant frozen in N2 and grind to fine powder. Work with dry ice or liquid N2.
50mg powder + 200uL Extraction buffer (50mM Tris/4% SDS/50mM DTT and protease inhibitors);
Incubate 30min at 4°C with shaking
centrifuge 15min. by 16`000xg at 4°C
Collect Supernatant
Precipitate with 2 Volumes of 10% TCA/0.07% DTT in acetone for at least 2 hr or longer
Centrifuge 15min. by 16`000xg and 4°C
wash TCA from your pellet by adding 2 Volumes of 0.07% DTT in acetone vortex well and repeat centrifugation and washin step at least 4 times
The final pellet should be white in collor (dry 15min. at RT)
Now you can reconstitute the pellet in every buffer that you want and need for 2D separation.
Regards
Natasha
Hi Prem,
Yes definitely phenol hinder during IEF. Please follow the protocol as mentioned by Natasha. Or you can follow
Plant frozen in N2 and grind to fine powder. Work with dry ice or liquid N2.
50mg powder + 200uL Extraction buffer (50mM Tris/4% SDS/50mM DTT and protease inhibitors).
Crush in liquid nitrogen several times at least 4-5 times. After that you can follow the centrifugation at 10000rpm for 45 minutes at 4 degree.
After that you may proceed with supernatant by using 2D clean kit provided by GE/G-bioscience to get unhindered IEF.
If further query follow papers.
best
Prashant
Hello
Phenol extraction method is the best method. But, from which part of the plant you want to extract protein ?? Accordingly plan your strategy. for example. plants seeds are rich in lipids and phytochemicals. First try to get rid of this.
Thanks
For protein extraction from plant tissue, the phenol protocol Works well. The color may be due to the presence of phenolic compounds and the pH. Check it. The quality of your simple can be assesed by a simple one-D SDS-PAGE. You will probably find that is dirty. If so, include one more step of clening by for example amonium acetate-metanol. .
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques