I have been trying this for more than 2 months, but it is always the same result. I am doing Fasciola hepatica whole cell lysate like below:
Adult parasites were homogenized in 10 mM Tris–HCl, pH
7.2 containing 150 mM NaCl, 0.5% Triton-X100, 1 mM
EDTA, 1 mM PMSF and 1 tablet complete Lysis protease inhibitor. To remove cellular debris, thehomogenates were solubilized for 30 min at 37C, followed
by centrifugation at 12,000g for 15 min at 4C.
When I do Ponceau S staining I do not see any higher bands than 40kDa. I load about 15-20 ug per well, but it did not improve until now.
Hi there,
Why is there a difference of staining color between HEK and FL samples? Do you use the same extraction protocol and loading buffer for the 3 samples?
For some reasons, HMW proteins may stay stuck in insoluble fraction or may aggregate in the upper gel or well during electrophoresis... Have you checked by running insoluble fraction and by transferring both stacking and separating gels?
Are you doing semidry or full-wet blots? In our experience, switching to tank from semidry can increase blotting efficiency for larger proteins significantly.
Hi again.
Thanks a lot Dominique.
I used a different lysis buffer for HEK cells because there is an already established protocol for them.
ð Place Cells on ice
ð Wash 3x with ice-cold PBS
ð Add 1mL RIPA buffer+protease inhibitor (500µl for 10cm dish)
ð Incubate for 15min on 4°C (shaker)
ð Harvest cells by scraping into 1.5mL Eppendorf tube
ð Centrifuge: 4°C; 13.000rpm; 10 min
ð Collect supernatant
RIPA Buffer:
10mM Tris (pH=7,4)
150mM NaCl2
1mM EDTA
1% Triton X-100
0,1% SDS
However loading buffer were same for all three samples, I checked stacking gel, it was OK, nothing seems to be aggregated there.
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