A protein whose pI is 8.9 - SDS PAGE (Mar/05/2006 )

Hi.

SDS page is wierd.

My purified His-tagged protein was migrated narrowed, has a vertical streak with a lot of protein remained in the well.

I don't think it's not the salt, or the gel condition because other protein samples loaded on the same gel migrated perfectly.

The questioned protein has its pI at pH 8.9, so I doubt the protein is aggregated on the SDS-PAGE.

Is anybody sure of possiblity, and if it's true, how can I solve the problem? Is there any way to see the basic protein on the gel?

Thanks in advance.

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sounds to me like chromatin is the problem. make sure to spin your sample like hell before loading it on a gel. also, make sure each sample is prepared in exactly the same method. this will tell you if your expressed protein is causing the aggregation. it could be that you have a highly insoluble protein thats causing aggregates to form. try loading less and do a western

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Thanks.

But I couldn't see any precipiation when the protein was stored at 4 C for one week.

I'll try anyway.

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We have worked on RNA and DNA binding proteins with similar or higher pI and they run fine in an SDS-PAGE gel. The band will sometimes run at an anomalous size but you probably need a higher pI than 8.9 for that to occur. How did you purify the protein? I know you used an IMAC column but did you use PEI to remove the nucleic acids? How is the A260 reading? How much salt did you use? Is the imidizole still present or did you exchange the buffer? Can you run a gel filtration column to further purify your sample? Just a few things off the top of my head.

dave

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hi,
in first instance, i would say that when u boil ur protein with SDS loading dye it will denature the protein and give completely negitive charge to your protein (coz SDS sits inbetween amino acids).
in this step your protein is completely -vely chareged. so i do not think that your protein will be retarded in the well itself because of pI, unless there is protein aggrigation or formation of macromolecules(complex with nucleic acids) by which ur protein cant run through the gel.

in second instance, u no need to worry about pI of your protein (8.9), coz stacking gel has pH of 6.8. if you say that u r proteins are not coming out of the wells means, some thing is worng with the protein form rather the pH.

coz of above reasons i wud say,
1, first have measurments of nucleic acids at 260nm in your protein as one of our member says,
2, make sure that your protein is not getting aggregated
3, if u get an idea about above two points and if you think both check points are fine, then look for information where your protein has some strange behavior at pH 6.8 (stacking gel) or at pH 8.8 (resolving gel).

hey
u r comments will be appreciated in regard to my way of above analysis

thanks
gud luck

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