I would prefer a ''home made'' technique rather than a kit suggestion! Thanks!
Well , you can try the same antigen recovery protocol as same as a immunohistochemical protocol using a citrate buffer an example of this is the following protocol.
After that proceed to the typical protein extraction from tissues using RIPA or NP-40 buffer with sonication.
1. Add 1ml ice-cold RIPA Lysis & Extraction Buffer to every 100mg o mammalian tissue.
2. Sonicate the tissue on ice with ~5 x 30 second at 50% pulse. Allow sample to cool between each sonication burst. Ensure tissue is completely homogenized before proceeding.
3. Incubate on ice for 5-15 minutes with periodical pipetting.
4. Centrifuge at ~14,000xg for 15 minutes to pellet cell debris. Transfer supernatant to a fresh tube for downstream applications.
Solutions and Reagents:
Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0):
Tri-sodium citrate (dihydrate) --------- 2.94 g
Distilled water --------------------------- 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.
Note: this buffer is commonly used and works perfectly with many antibodies. It gives very nice intense staining with very low background.
Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0):
Citric acid (anhydrous) --------------- 1.92 g
Distilled water -------------------------- 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N NaOH and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.
RIPA buffer
10 mM Tris-Cl (pH 8.0)
1 mM EDTA
0.5 mM EGTA
1% Triton X-100
0.1% sodium deoxycholate
0.1% SDS
140 mM NaCl
The above solution is stable at room temperature.
Before use, add:
1 mM PMSF
http://www.ihcworld.com/_protocols/epitope_retrieval/citrate_buffer.htm
http://cshprotocols.cshlp.org/content/2006/4/pdb.rec10617.short
https://www.gbiosciences.com/image/pdfs/protocol/786-489_protocol.pdf
Hi, Marie.
It may not be possible or may lose many proteins that became insoluble in the fixation procedures. However, try the followings.
1) Gradual rehydration of the tissues in descending % (90, 70, 50, 20 and 10) of EtOH. Give enough time (`30 min each) to release and replace the EtOH, depending on the size of tissues (10mm3 as a standard).
2) Wash tissues in enough amount of ddH2O at the final stage (3x washes for 1h each) to remove remaining aldehydes. Possiblly O/N with gentle rocking.
3) Blot the tissues to drain H2O.
4) Plunge the tissues into a separate container filled with LN2 (-196C).
5) Transfer tissues into cold mortar and pestle (previously cooled -70C over hours).
6) Pour LN2 into the tissue in the mortar.
7) Grind the tissues completely while pouring additional LN2.
8) Collect the powdered tissues into several 1.5ml eppendorf tubes containing lysis buffer.
9) Rock the tubes at 4C for ON. Spin it to collect supernatant, Measure protein contents.
10) 2x SDS sample buffer to dissolve proteins and spin, collect supernatant for SDS-PAGE.
Good luck
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