protein truncation with chemiluminescent detection

Stephen Dahl stayve-and-irayne at worldnet.att.net
Sat Oct 21 16:13:17 EST 2000


Using the TNT T7 version kit we've had excellent and not so excellent
results.  Parameters that appear to have an effect include the preparation
(some preps work great, some not so great. Not sure why, but a few extra P/C
extractions can sometimes raise a given prep's abilities), the presentation
(linear or circular) and the sequence of the DNA.  I've found it a fussy,
but useful kit.  I sure as heck wouldn't use a secondary detectiion system
though.  35s-methionine or cysteine would be the way to go in my opinion.


"Ruth A Dworak" <Ruth.A.Dworak at uth.tmc.edu> wrote in message
news:l03130301b616065eb8df@[129.106.39.172]...
> I am a PHD student, and I am currently trying to ultimately do a protein
> truncation test over 5000 bp of an exon.  We are trying to accomplish this
> in two 3 KB pieces.  As of yet, we are just trying to get the first 2000
> basepairs of the exon working with the transcription/translation system.
>
> I am using Promega's TNT Quick Transcription/Translation System, product
> #L1170,
> and Promega Transcend Non-Radioactive Translation Detection System
> (chemiluminescent version), product #L5080.
>
> In my reaction, the homozygote truncated sample showed a strong clear
band,
> and heterozygote showed a faintly detectable band for the truncated
protein.
> There were no clearly detectable bands for either the homozygote normal or
> the heterozygote non-truncated protein.  There was no overall background
on
> the membrane, but "background" bands did appear in all samples, including
a
> negative control.  Several of these background bands appear at around the
> size of the expected nontruncated protein that I am having trouble
> definitively detecting.
>
> I would greatly appreciate any suggestions for others who have have
> experience with this system or similar systems.  I am guessing that my
> problem lies in too low a yield from the transcription/translation
> reactions, resulting in me having to have longer exposure times which also
> show these interferring background bands.  Do you know which components of
> the reaction I can alter trying to optimize the reaction?  Alternatively,
> does anyone know if unspecific binding of Streptavidin-HRP to other
> proteins instead of the biotin-labeled lysines could be the problem?
>
> Thank you for your time and help.
>
> Ruth Ann
>
>
>
> ---







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