protein truncation with chemiluminescent detection

[Ruth A Dworak]

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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