Re: Prot Seq: when isn't it logical to assume a 3 times load

Tim Slattery (Tim_Slattery@berlex.com)
Mon, 22 Sep 1997 09:04:40 -0700

Tom,

In my experience it doesn't work that way; using two lanes of equal
concentration of protein on PVDF does not give a 2x signal compared to
one lane. Doubling the amount of protein blotted on to a fixed amount
of PVDF comes much closer to doubling the signal.

Minimizing the amount of PVDF loaded in to the sequencer will give
lower background and more signal compared to an equivelent amount of
protein loaded in the presence of excess PVDF. Since blotted bands
rarely saturate the PVDF adding multiple bands I have assumed is
equivelent to adding some unblotted PVDF, reducing the signal and
adding to the background. Why at some point during the sequencing the
signal would suddenly jump to a higher, however, is beyond me.

Tim Slattery
Protein Biochemistry and Biophysics Department
Berlex Biosciences
tim_slattery@berlex.com


______________________________ Reply Separator _________________________________
Subject: Prot Seq: when isn't it logical to assume a 3 times load?
Author: Thomas J Miller E328/247 695-1745 <MILLERTJ@ESVAX-A1.EMAIL.DUPONT.COM>
at Internet
Date: 9/19/97 9:53 AM

Hello fellow sequencers. I just had an experience where it was safe (in my
mind) to assume that by tripling a pvdf sample load from 2 to 6 tracks, I
would see a likewise triple initial yield. Well I didn't. I saw a doubling
of the yields in the first half of the sequencer run, then strangely,
the expected 3x amounts. The sample origins was an overexpression in E-coli.
The pvdf blot looked consistent across the sheet. I'm using a Beckman
LF3000 sequencer. Any comments or suggestions as to why I saw this effect
would be appreciated. Thank you. Regards, Tom

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Thomas Miller, Macromolecular Analysis, Protein Sequencing
Dupont Co., POB 80328, Wilmington, DE 19880
Voice 302-695-1745
email: Thomas.J.Miller@usa.dupont.com
Opinions are my own and not of my employer.
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