1. Western Blot 200k 68k 100k 43k MW

2. Lysis Buffer Lysis Lyse tissue. This requires a lysis buffer: 50mM Tris-HCL (ph7.4), 150mM NaCL, 1% Triton x100 and protease inhibitors. To make up 30ml of lysis buffer do as follows: 1.5ml of 1M Tris-HCL 4.5ml of 1M NaCL 300 ul of Triton. (Add this last and dissolve using hot water) Place lysis buffer on ice. All lysis steps should be done at 4 degrees C. Add requisite amount of protease inhibitors. Add 1ml of lysis buffer to large brain regions such as the cortex. Add 100ul to regions such as the hypothalamus, or superior colliculus. Most brain regions can be lysed in 1ml. Homogenise samples. Sample should be kept on ice as far as possible. This can be done with either a pellet pessel or a homogenised. All tissue to lyse for 1hour on ice. Vortex intermittently. Centrifuge at 20,000g (or max) for 30mins at 4 degrees. Take off supernatant, avoiding particles, and pellet.

3. Lysis Buffers PBS Triton X-100 High salt: 300 mM NaCl Deoxycholate

4. Western Blot Lysis Buffer 1% SDS, 1 mM EDTA, 10 mM Tris/HCl pH 7.4, 90 o C. Virtually 100% solubilization Rapid stop of virtually all proteolysis and phosphorylation/dephosphorylation Suitable for most protein assays

5. Western Blot 200k 68k 100k 43k MW

6. Western Blot http://www.ideascientific.com/genie.htm GENIE® Electrophoretic Transfer

7. Blocking Buffers Carnation Instant Milk –Works better than generic powdered milk –Cannot be used for global or site specific P- tyrosine antibodies due to high basal tyrosine phosphorylation of the milk

8. Western Blot 200k 68k 100k 43k MW

9. Antibody Selection Commercial antibodies –Very few companies manufacture their own antibodies or have any semblance of QC –Many companies sell exactly the same antibody (check buffer and PDS for clues) –Some very good antibodies may simply not work in your hands –The lower the concentration of a polyclonal antibody the better if all else is equal –Recognition of a single band not required –It is a myth that monoclonal antibodies are more specific than polyclonal antibodies Grow Your Own

10. Western Blot 200k 68k 100k 43k MW

11. Western Blot Quantitation C Tx IR Units 1450 1500

12. Western Blot Quantitation

13. Standard CurveExperimental Samples