Above answer are more than enough for the extraction purpose but have a look at this too
Article Functional expression of the taste-modifying protein, miracu...
Hello Fakhria.
Any protocols would be fine for protein extraction and subsequent Western blot if you follow the suggestion;
Chop the leaves into small pieces (2~3mm in size) with a pair of sterile surgical scissors.
Plunge the pieces of leaves into LN2 gas, store at -70-~80C until use.
Add an appropriate amount of pieces into a cold mortar and a pestle that previously stored at -70C. Grind the leaves into powder, if necessary pour some LN2 gas. This powder can be stored in Eppen tubes for later analysis.
Start protein extraction with RIPA buffer with proteinase inhibitor mixture.
Rock the tubes at 4C for 2h to dissolve proteins in maximum.
Protein content analysis.
Mix it with 2xSDS sample buffer, followed by rocking it at 4C for 1h.
Follow the routine for SDS-PAGE, etc.
You will never fail if you take care of critical steps seriously.
Best wishes.
Dear Dr. Fakhria
Solution ; SDS-Tris-Urea extraction buffer (100 ml)
1- Take 5 gram of leaves after grinding placed into Eppendorf tube ( 1.5 mL)
2- Add 1 mL of solution the sample were mixed for 5 min followed by incubation for 2 hours at 20 C .
3- The mix was centrifuged at 20 C at 4000 rpm for 20 min .
4- The supernatant was collected and stored ( _ 20 C ).
Best regards
Hi,
the easiest way is generally to directly grind the tissue in denaturing sample buffer (Laemmli buffer or comparable denaturing sample buffer fitting your gel system). Then, boil for a few minutes (e.g. 3-5 min @ 95°C, or 10 min @ 70°C), spin briefly to sediment tissue remnants, and load on gel. It will depend on your plant species whether the tissue will be "soft" enough for grinding in a tube with a pestle (https://www.sigmaaldrich.com/catalog/product/sial/sial501zz0?lang=de®ion=DE) - you don't give that kind of information in your question. Otherwise, this is very reliable as it is a single step and extremely simple. In respect to w/v, 2 volumes of sample buffer are a good starting point, and loading ~ 10 µl of this will be fine for SDS-PAGE and western blotting in most cases.
My plant specie is lettuce. Is this like extraction and sample preparation in a single step? Can you please send me the detailed protocol for this method of protein extraction. Thanks.
Above answer are more than enough for the extraction purpose but have a look at this too
Article Functional expression of the taste-modifying protein, miracu...
Hi. Dr. Fakhria
Greetings
For Rapid isolation of protein for SDS-PAGE analysis according by Hames and Rickwood (1990):
One gram of tomato samples was placed in a mortar and pestle and added 2ml of QB-buffer the components of QB –buffer (100ml) was prepared as follow:
o 2M KPO4 (pH 7.8) 5ml 100mM
o 0.5M EDTA 200μL 1mM
o Triton X-100 1ml 1%
o 80% Glycerol 12.5ml 10%
o Distilled, sterile water 81.1ml
o DTT (1M) 100μL 1mM (Alternatively directly add 15.4mg DTT per 100ml) was added immediately before using.
o QB w/DTT was stored at –20°C.
The liquid grindate were removed into a microfuge tube and placed on ice. The samples were centrifuged at 15,000rpm for 15 minutes at 4°C. The liquid supernatant was transferred into new eppendorf. Then the samples were stored in -80°C.
Good Luck
A protocol:
- harvest 50 mg of lettuce leaf tissue in a 1.5 ml tube
- add 100 mikroliters of 2 x laemmli buffer
- grind tissue with micropestle
(If too dense, you might have to add another 50 microlitres of buffer)
- boil 5 min 92 degree Celsius
- spin 30 seconds full speed
- load 10 microlitres on a gel
Do western / coomassie / whatever
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