Aggregated samples at the top of SDS-PAGE gel - (Apr/28/2010 )
My problem is as follows:
i've made several western blot! i saw frequently huged band over 250kda ( molecular weight not expected for my protein of interest).
i made w.b today, i had the idea of the stucking gel that was in small area, after the polymerization of the gel, i could remark that stucking part is not large, the samples loaded into the wells have few space in the stucking gel!!!
may be it's for this reason that in the western, i saw the band of my membrane protein of interest, but also huged band sticked above !!!
Is it due to the stucking gel?????????
thanks for your replies and ideas
what is your sample? Eukaryotic cell lysate? what is sticked? stucking gel? you mean stacking gel?...guess you have problem with stick...lol....
I didn't understand, your problem is on the gel or the membrane? the topic says sds-page, but inside the thread you say western blot.
how do you prepare you cell lysate? the membrane precipitation is the last part of cell fractionation step and it needs very high speed. so most probably it is not membrane.
Curtis on Apr 29 2010, 12:39 PM said:
I didn't understand, your problem is on the gel or the membrane? the topic says sds-page, but inside the thread you say western blot.
how do you prepare you cell lysate? the membrane precipitation is the last part of cell fractionation step and it needs very high speed. so most probably it is not membrane.
my sample is total cell lysate (brain). in fact, what i means by sticked is on the western blot, i see in the filter huged band over 250kda! plus the band of my protein of interest at 150kda. so, im wondering if the upper band is due to the piece of stucking that remains with resolving gel and was transferred as well!!??
For sds-page, the stucking gel covering the wells and not large space for the stucking.
i hope ou understand me!
thanks
luciana on Apr 29 2010, 06:05 AM said:
Curtis on Apr 29 2010, 12:39 PM said:
I didn't understand, your problem is on the gel or the membrane? the topic says sds-page, but inside the thread you say western blot.
how do you prepare you cell lysate? the membrane precipitation is the last part of cell fractionation step and it needs very high speed. so most probably it is not membrane.
my sample is total cell lysate (brain). in fact, what i means by sticked is on the western blot, i see in the filter huged band over 250kda! plus the band of my protein of interest at 150kda. so, im wondering if the upper band is due to the piece of stucking that remains with resolving gel and was transferred as well!!??
For sds-page, the stucking gel covering the wells and not large space for the stucking.
i hope ou understand me!
thanks
Are you using reducing condition?