how to deal with protein degradation - (Nov/20/2007 )
i never performed cosedimentation assays with the myosin and f-actin, just atpase activation assays (this is not entirely true, we did prepare samples of myosin or hmm or s-1 with f-actin for electron microscopy but were not concerned with efficiency).
but...
i would be concerned that the salt concentration is a little low to maintain the actin in the filamentous form. we always made sure there was at least 0.1M kcl (if not 0.15M). this allows the actin and the myosin to remain filamentous.
by the way, how do you judge that the f-actin is 4uM? have you determined the (average) size of the filament? do you base the concentration on the g-actin before polymerizing? we always considered the concentration as ug/ml.
-mdfenko-
QUOTE (mdfenko @ Mar 28 2008, 07:02 AM)
i never performed cosedimentation assays with the myosin and f-actin, just atpase activation assays (this is not entirely true, we did prepare samples of myosin or hmm or s-1 with f-actin for electron microscopy but were not concerned with efficiency).
but...
i would be concerned that the salt concentration is a little low to maintain the actin in the filamentous form. we always made sure there was at least 0.1M kcl (if not 0.15M). this allows the actin and the myosin to remain filamentous.
by the way, how do you judge that the f-actin is 4uM? have you determined the (average) size of the filament? do you base the concentration on the g-actin before polymerizing? we always considered the concentration as ug/ml.
but...
i would be concerned that the salt concentration is a little low to maintain the actin in the filamentous form. we always made sure there was at least 0.1M kcl (if not 0.15M). this allows the actin and the myosin to remain filamentous.
by the way, how do you judge that the f-actin is 4uM? have you determined the (average) size of the filament? do you base the concentration on the g-actin before polymerizing? we always considered the concentration as ug/ml.
yes, the 4uM is just base the concentration on the g-actin before polymerizing. my former colleague told me to calculate like that.
what kind of buffer do you use for actin polymerization? we polymerize g-actin at 10mM Hepes, 30mM KCl, 2 mM MgCl2 1mM 2-mercaptoethanol and 2mM NaN3. some literatures suggested ATP was added. i don't know whether it is really needed. from what i observed, our F-actin was short, most of them were shorter than 1 cm. i have no idea if it is because of the ATP or the low salt concentration.
anyway, i would like to try higher salt concentration as you suggested.
thanks a lot
-lical208-
QUOTE (lical208 @ Mar 31 2008, 09:53 AM)
yes, the 4uM is just base the concentration on the g-actin before polymerizing. my former colleague told me to calculate like that.
what kind of buffer do you use for actin polymerization? we polymerize g-actin at 10mM Hepes, 30mM KCl, 2 mM MgCl2 1mM 2-mercaptoethanol and 2mM NaN3. some literatures suggested ATP was added. i don't know whether it is really needed. from what i observed, our F-actin was short, most of them were shorter than 1 cm. i have no idea if it is because of the ATP or the low salt concentration.
anyway, i would like to try higher salt concentration as you suggested.
thanks a lot
what kind of buffer do you use for actin polymerization? we polymerize g-actin at 10mM Hepes, 30mM KCl, 2 mM MgCl2 1mM 2-mercaptoethanol and 2mM NaN3. some literatures suggested ATP was added. i don't know whether it is really needed. from what i observed, our F-actin was short, most of them were shorter than 1 cm. i have no idea if it is because of the ATP or the low salt concentration.
anyway, i would like to try higher salt concentration as you suggested.
thanks a lot
our g-actin is in a buffer that contains atp (to maintain the g-form), 2mM tris, 0.2mM atp, 0.2mM cacl2 and 0.5mM 2-me, pH 8.
we added mgcl2 to 1mM and kcl to 100mM then incubate at 37C. spin at 140 000xg for 1 hour to collect. suspend in 100mM kcl and 50mM tris, pH 8 (pH and concentration of buffer depends on the use).
after suspension, i determined the protein concentration (mg/ml).
-mdfenko-