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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. •  

Lysis Buffers

• PBS

• Triton X-100

• High salt: 300 mM NaCl

• Deoxycholate

Western Blot Lysis Buffer

• 1% SDS, 1 mM EDTA, 10 mM Tris/HCl pH 7.4, 90o C.

• Virtually 100% solubilization

• Rapid stop of virtually all proteolysis and phosphorylation/dephosphorylation

• Suitable for most protein assays

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Western Blot

http://www.ideascientific.com/genie.htm

GENIE® Electrophoretic Transfer

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

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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

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Western Blot QuantitationC Tx

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Western Blot Quantitation

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