Hi All
Could anyone share with me the protocol they follow to do western blotting for adult worm samples? Presently, I am thinking of boiling 100 ul of worms in Laemmili buffer with beta mercaptoethanol and protease inhibitor added to the buffer. Is this correct?
Any comments/insight would be helpful as I am doing this for first time.
Thanks
Joan
jmg326
August 17, 2012, 11:07pm
2
Harvest about 500 microlitres to 1 ml of animals in a 15 ml conical tube.
Add lysis buffer:
KCl 150 nM
EDTA 1 mM
SDS 0.25%
NP-40 1.0%
Tris/HCl pH 7.4 50 mM
Protease inhibitor (e.g. PMSF)
Sonicate to disrupt cuticle: 3x20 pulses, 35% output using microtip. Chill on ice between sets.
Transfer to 1.5 ml tube, spin down 20,000g, 15 min, 4 degrees C.
Keep the supernatant (has your protein extract in it) and discard pellet.
Worm Western Lysis Buffer:
100mM Tris pH6.8
8% SDS
20mM BME (~1%)
Recipe: total 1ml
1M Tris 100ul
10%SDS 800ul
BME 10ul
dH2O 90ul
5ul lysis buffer into PCR tube lid
hand pick 20 animals into buffer (avoid bacteria);
Close Lid and spin sample to bottom;
100°C for 5 min in PCR machine (or 37°C for 30-60 min for multipass membrane proteins like SMF-3 and MIG-14), 4°C cool down and hold;
5ul 2xSDS loading buffer with dyes;
Load SDS-PAGE gel;
Notes:
Often use 10ul with 40 animals to load thicker 1.5mm gel.
anti-GFP 1:3000 in 1% milk 4°C overnight, after 5% milk blocking RT 1 hour.
HRP-conjugated Goat anti-GFP antibody
(http://www.fitzgerald-fii.com/gfp-antibody-hrp-60r-gg002hrp.html ) ; cat#RDI-GRNFP3abg-HRP, new cat#: 60R-GG002hrp