[RASMB] Compare MW obtained from Segal vs. Origin

[Chin, Christopher]

Dear Ariel and colleagues,

First let me thank you for your response to my inquiry.

Maybe I am naïve, without trying to interpret the result obtained from the
equilibrium curve (John Philo is correct, after 4days of tryptic digestion
with this protein complex at 37C, some proteolysis does occur), I was only
use this equilibrium curve (or any curve), to compare MW obtain from these
two software, I thought by inputting the raw data using exactly the same
window (radius boundary), I should be getting the same empirical number
(MW), or close to it. Maybe I am off base on my reasoning.  

As to solution column and time of equilibrium issue, using the standard
cell, the maximum volume one can load is about 0.45ml. In most of my
experiment, I try to shot two birds with one stone by loading about 0.2ml to
do a SV run (3-5 hours), after collecting enough scans I then reduce the
speed and reprogram for SE run (20-24 hours). I have no problem with it if
the protein is stable. By doing scan subtraction at every two hours, I found
that for 0.2ml sample, 20-24 hours is sufficient for (stable) sample to
reach equilibrium. However I found that for the radius region above 7.1,
near the base of the cell, apparent due to reflections, as John Philo point
out, it will never reach zero on scan subtraction no matter how long I
continue to spin it so I try not to use the data points beyond 7.1. 

I agree with John Philo's point

"Knowing what is 'good' or 'bad' data in equilibrium experiments is
unfortunately a bit of an art"<?xml:namespace prefix = o ns =
"urn:schemas-microsoft-com:office:office" />

I still remember a long while back, taking picture with a camera use to be
an art. One needs to know how to set f value, how to estimate distance as
well as aperture.  Expert and experience user takes clear picture while
amateur takes blurred picture. But now a day you can take decent picture
just with a disposable camera. Back to AUC, I was hoping I could combine
Segal and Origin to enhance my betting average of picking up the best subset
to use.

 I think it is cool to be able to copy the whole page information.  I hope
Segal Program Can provide a "page copy" function, similar to the Beckman
Origin, so page data can be copy and paste to Microsoft word for record
keeping. I saw and like the page information you sent. I could not figure
out how to do it.  I finally realize that the page you sent is in ".jpg
file", which I believe you have to scan with Digital camera. It would be
very nice if we can just copy the page, like the Beckman Origin does, and
paste the finished page to the Microsoft word for record keeping. At the
present time I could only copy and paste each figure for record keeping,
very time consuming.

I have since test another set of equilibrium curve. It is a pure protein. It
is a self-associated system.  I am please to see these results. I also post
the raw data 00005.ra1 for anyone interest in using it for practice.

I was wondering because this curve seems agree better with the previous
curve, is this means that these software are system dependent.  I mean if
the system is a self associated system, both software will converge to
pretty much the same answer.  If the system has other low mass contaminate
(or even some proteolysis), then the answer will not always be the same even
using the exact same window or radius region.  This is only my wild guess.

 

Regards,

 

Chris

Summary: Compare 4 edited subset data from raw data 00005.ra1 using Origin
vs.. Segal.

 


Edit Equilibrium curve

Boundary radius region used

MW obtained from Origin / variance

(Confidence limit)

MW obtained from Segal / correlation

 


Discrepancy@ %



Edit 1

6.806-7.112

82KD / 11.8

(79-84KD)

82 KD / 0.999876

0


Edit 2

6.685-6.984

86KD / 4.6

(83-89KD)

87 KD / 0.999972

-1.2


Edit 3

6.831-7.076

81KD / 11.6

(77-84KD)

83KD / 0.999018

        -2.5


Edit 4

6.689-6.975

89KD / 4.1

(85-92KD)

89KD / 0.999959

0

@ (MW obtained from Origin  -  MW obtained from Segal) / MW obtained from
Origin 

Using default rho(1.006) and v-bar(0.73) for comparison. 

Use default V-bar=0.73 , rho=1.006

 

 

 

 

 

-------------------------------------------------------------------------- 
Christopher Chin 
Manager, XLA-Analytical Ultracentrifugation facility 
Sealy Center for Structural Biology 
HBC&G, 5.134 MRB.UTMB, Galveston,Tx 77555-1055 
cchin at utmb.edu, 409-772-1693, efax 708-585-1920 
--------------------------------------------------------------------------- 


 
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