Jhdavis::Western::Eyal

running the western

soak gel in cold 1x transfer 5 min, wet PVDF membrane in MeOH then soak along with blotting paper in 1x transfer
assemble:
--anode (red)--
--blotting paper--
--blotting paper--
--PVDF--
--gel--
--blotting paper--
--blotting paper--
--cathode (black)--

transfer at 1mA/cm2 for 1 hour
10x Transfer Buffer:
250 mM Tris
1.92 M Glycine
mixture pHs itself to pH 8.5

1x Transfer Buff:
80 mL 10x
720 mL H20
200 mL Methanol

10x PBS (1L):
20 g NaCl (344 mM)
2 g KCl (27 mM)
14 g Na2HPO4
2 g KH2PO4

1x PBS-tween:
1x PBS
Tween20 at 2 mL/L

Blocking Buffer:
5% Milk
0.02% Na-Azide
PBS-Tween

Washing:
PBS-Tween

Protocol:
1 hour block
add primary 1:10,000 o/n
3x wash
1 hour with secondary 1:10,000
4x wash
ECL exposure

for ecf, use these setting on the typhoon: add 1 mL substrate (mixed powder/solution, saved at -80, save thawed at 4 for 2-4weekds expose 5-20 mins, place protein side down in typhoon, Focal Plane – Platen Pixel Size – 200 μ Sensitivity – Normal Table 3. Instrument settings for imaging fluorescent Western blots. Chemifluorescence method Substrate (excitation, emission maxima) Laser Emission filter ECL Plus (430 nm, 503 nm) 457 nm* 520BP40* ECF (440 nm, 560 nm) 532 nm 526SP† DDAO phosphate (646 nm, 660 nm) 633 nm 670BP30 Direct fluorescence method Fluorochrome (excitation, emission maxima) Laser Emission filter Fluorescein (495 nm, 520 nm) 532 nm 526SP† Cy3 (550 nm, 570 nm) 532 nm 580BP30 Cy5 (649 nm, 670 nm) 633 nm 670BP30

use 532, 526SP