RIPA
150 mM NaCl
1 mM PMSF
1 mM EDTA
5 µg/ml Aprotinin
5 µg/ml Leupeptin
1% Triton x-100
1% Sodium deoxycholate
0.1% SDS
50 mM Tris, pH 7.4
NP-40
NaCl 150 mM
NP-40 1%
Tris, pH 8.0 50 mM
HEPES
Hepes pH 7.4 50mM
NaCl 150mM
Glycerol 10%
Triton X-100 1%
KCl 5mM
EDTA 1mM
Protease inhibitors (leupeptin 10ug/ul, aprotinin 10ug/ul, PMSF 1mM)
Phosphatase inhibitors (NaF 50mM, Na3VO4 0.5mM, Na4P2O7 5mM)
To prepare 1 liter of 10X TBS:
30 gr Tris
88 gr NaCl
2 gr KCl
mix everything in dwater until it’s disolved. Adjust pH at 7.5
To prepare 100ml of PFA 4% stock solution:
- Add 86ml ddwater in an enlermeyer
- Boil water in the microwave
- Weigh 4g of PFA
From now on in the fume hood
- Transfer PFA in the erlenmeyer
- Add a clean stirring bar
- Add a drop of NaOH 10M
- Stir it and keep it hot (heat at #1-2) till it goes [...]
A complete western blot protocol from Howel lab in University of California San Diego. Here is the link:
http://cancer.ucsd.edu/howelllab/Western.html
From how to obtain a cell lysate to how to prepare the gels, running the gels, membrane transfer, detection and stripping the membrane. Also all the reagents and buffers you need for your western blot.
There is many techniques, this [...]
Laemmli is a sample buffer to use in western blot. 6X sample buffer is added to each protein sample and is boiled or heated for 5-10 minutes.
Recipe to prepare 10 ml:
- 1.2gr SDS (sodium dodecyl sulfate)
- 6mg bromophenol blue
- 4.7ml glycerol
- 1.2ml Tris 0.5M pH6.8
- 2.1ml dwater
warm it a little bit and shake it till [...]