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In Topic: Transcription/Translation reaction not resolving
04 June 2013 - 11:31 AM
I figured it out. By loading 1/3 the amount of translation reaction and using LDS sample buffer with NuPAGE gels, the gels look very nice. The biggest thing was the overloading. With a TnT reaction, I assume there are a lot of components that interfere with resolving a gel.
In Topic: Protein amount to load for publication figure?
11 May 2013 - 07:02 PM
Thanks for the suggestions. I will be coomassie staining on a 1mm gel. The sizes are 20 & 70 KDa. I will try the 2ug and see how that goes
In Topic: break disulfide bonds to study fluorescence
06 May 2013 - 09:25 AM
use DTT instead of 2-mercaptoethanol. DTT will break the disulfide bonds just as well.
In Topic: trouble in his-tagged protein binding with Ni-NTA agarose
02 May 2013 - 10:47 AM
Oh OK, I was definitely confused with your question. "so i am losing my protein sample" threw me off. It seems that you have it figured out. The higher NaCl, the presence of tween and or/ tritonX-100 are causing this.
If you need to store your protein in that binding buffer with the extras, you can elute with the first buffer, and then dialyze with the second.
If you need to store your protein in that binding buffer with the extras, you can elute with the first buffer, and then dialyze with the second.
In Topic: Contaminants in protein purification.....help!
02 May 2013 - 10:41 AM
A couple of things...
To determine if they are degradation products (ie the 10kd) you can do a western blot and subsequent anti-his antibody. This will show what has the his-tag.The 50KDa band could be a dimer. I frequently observe dimers with high expression levels. The best way to remove these contaminants is by size exclusion chromatography.
A while back I was having the same problem. I started freezing the pellets before cell lysis, and I took great care (and time) to resuspend the pellet and lyse the cells. This better cell lysis led to the contaminant bands disappearing. Once I lysed the cells and did high-speed centrifigation, I hardly observed a pellet (cell wall, etc). This is a good indication that the lysis was done properly.
To determine if they are degradation products (ie the 10kd) you can do a western blot and subsequent anti-his antibody. This will show what has the his-tag.The 50KDa band could be a dimer. I frequently observe dimers with high expression levels. The best way to remove these contaminants is by size exclusion chromatography.
A while back I was having the same problem. I started freezing the pellets before cell lysis, and I took great care (and time) to resuspend the pellet and lyse the cells. This better cell lysis led to the contaminant bands disappearing. Once I lysed the cells and did high-speed centrifigation, I hardly observed a pellet (cell wall, etc). This is a good indication that the lysis was done properly.
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