tag:blogger.com,1999:blog-1195222727010537305.post7589850047248512744..comments2023-08-03T07:13:32.291-05:00Comments on Aptamer Project Site - 10% APS for Everyone: mCherry Fluorescent Protein Progress Report TwoAptamer Staffhttp://www.blogger.com/profile/11584531632318072295noreply@blogger.comBlogger3125tag:blogger.com,1999:blog-1195222727010537305.post-90438442096418989752011-10-25T11:13:05.148-05:002011-10-25T11:13:05.148-05:00Hi,
I am facing some problems with my Selection....Hi, <br /><br />I am facing some problems with my Selection. I had posted earlier in the start of the year that I am working on selecting an aptamer against Ebola Glycoprotein. About the glycoprotein -- Its a trimer and has a histidine tag. <br />I performed 9 rounds of selections including negative selection with a protein to remove sequences binding to His Tag and also competition assay with a protein receptor. After 9 rounds, I did filter binding assay to check the Kd and unfortunately I didn't see any binding at all. Everything was background. <br />What is interesting is that, before starting with selections I threw the starting pool, against the protein and got a 500nM Kd, suggesting that this protein might be a good target for selection. <br />But when I tried repeating the same experiment with starting pool I didn't see any binding. I concluded that the problem could be with my protein so I ran a native gel and surprisingly the protein too is intact. I am absolutely puzzled as to what could be the problem. <br /><br />Could you please suggest me some changes or any ideas as to what must be a problem here ?<br /><br />I would really appreciate any comments or suggestions.<br /><br />Thanks, Shambhavishambhavihttps://www.blogger.com/profile/01273283706084980844noreply@blogger.comtag:blogger.com,1999:blog-1195222727010537305.post-85365461432390494822011-01-22T01:00:24.378-06:002011-01-22T01:00:24.378-06:00Actually Alec's reasoning is a little misleadi...Actually Alec's reasoning is a little misleading. He is correct that it reduces background, but it does so not by binding the protein (target). The tRNA will bind to sites on the seiving matrix (the filter or the beads) that have a propensity to bind RNA nonspecifically. These sites could also bind the amplifiable pool, diluting out the actual target specific binders in the PCR amplification. The tRNA is added at 5x to 10x molar excess of the pool and strongly competes thermodynamically for the nonspecific binding sites on the filter or bead and cannot be further amplified in the PCR.Brad Hallhttps://www.blogger.com/profile/10516353314281930175noreply@blogger.comtag:blogger.com,1999:blog-1195222727010537305.post-31187462033461210512011-01-19T17:10:07.592-06:002011-01-19T17:10:07.592-06:00Hi,
I am a graduate student working on Selection...Hi, <br /><br />I am a graduate student working on Selection of aptamer against ebola virus. I was wondering why did you add tRNA in the binding buffer. It says to reduce background but can you elaborate more on it.<br /><br />Thanks, <br />Shambhavishambhavihttps://www.blogger.com/profile/01273283706084980844noreply@blogger.com