Pulse - Chase:

1) Add complete medium containing 10,000X excess cold methionine for the desired chase time. The chase time will determine which window of the biosynthetic process you will be examining.

i. 10 min label + 0 chase = initial product (e.g. non-glycosylated, precursor probably in sol fraction)

ii. 10 min label + 15-30min chase = sequential biosynthetic products eg.glycosylated then proteolytically cleaved forms

iii. 3hr label and 15-30min chase = final product + intermediates

iv. 3hr label + 1-2hr chase = final cleaved and glycosylated, mature protein probably in insoluble fraction

v. 3hr label + 0 chase = all stages in biosynthetic pathway

LYSIS

1) [PENN] 50l of Laemmli buffer (2% SDS, 62mM Tris pH 6.8, 10% glycerol) and boil for 5mins, shear and sonicate 2X. Dilute to 1ml in RIPA buffer (20X)to bring the SDS to 0.1%. Spin in eppendorf 15mins and discard any pellet.

2) TED/ERWIN Add 200l of lysis buffer to dish and scrape(25mM Tris pH 7.4, 3mM EDTA, 100mM NaCl, 2% SDS, 0.2% Trasylol, 1mg/ml BSA, + protease inhibitors=1mM DTT,0.5ug/ml Leupeptin, 1ug/ml leuleuleu)?sonicate 2X for15 secs stop 6 with 10sec on ice between. Boil 2mins Dilute with 1ml 2% triton,150mM NaCl, 50mM Tris pH 7.4, 1mM EDTA This brings the final SDS conc to below 0.5% SDS and complexes the SDS into micelles. Spin 15 mins in eppendorf.

 

3) RIPA SOLUBLE/INSOLUBLE FRACTIONS: (This is used to determine at what point proteins oligomerise or become attached to the cytoskeleton.) Add 300l-1ml/35mm dish of RIPA buffer to cells on dish leave on rocker in cold for 10mins. Scrape cells into eppendorf and spin 50,000g for 10 mins. Collect supn =soluble fraction. Process pellet as described above =insoluble fraction.

RIPA/WASH BUFFERS:

20mM Tris 7.5

0.15M NaCl

1mM EDTA

1% NP-40

If background is a problem include in washes

0.5M NaCl

0.5% deoxycholate

0.1% SDS =100mM Tris pH 6.8

NELSON extracts in300l/35mm dish for 10 mins 4C

0.5% Triton-X100

120mM NaCl

25mM KCl

2mM EGTA

2mM EDTA

0.1mM DTT

1mM PMSF

15mM Tris pH 7.5

Final washes are 4-5 times in

1% Triton final conc.

5mM EDTA final conc.

1% deoxycholate

0.1% SDS

0.1mM DTT

1M NaCl and 1 final wash in TE +0.5mM DTT

ERWIN TED NELSON PENN

LABEL: 250 250 250/500 250

LYSE 2% SDS 1% SDS 0.5% Triton 2% SDS

PRECIP 0.66% SDS 0.16% SDS 0% 0.1% SDS

1.25% Triton 2.0% Triton 0.5% Triton 1% NP-40

WASH 0.2% SDS 0% 0.1% SDS 0.1% SDS

1% Triton 2% Triton 1% Triton 1% NP-40

1% Deoxychol. 0.5%deoxyc

150mM NaCl 1M NaCl 0.5M NaCl