Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > General Science Forums > Biochemistry Forum
Register Search Today's Posts Mark Forums Read

Biochemistry Forum Discuss and post questions regarding the study of Biochemistry. If you need homework help this is the place to ask!


spectrophotometry enzyme substrate dilution problem

spectrophotometry enzyme substrate dilution problem - Biochemistry Forum

spectrophotometry enzyme substrate dilution problem - Discuss and post questions regarding the study of Biochemistry. If you need homework help this is the place to ask!


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 02-21-2009, 11:44 AM
Pipette Filler
Points: 10, Level: 1 Points: 10, Level: 1 Points: 10, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default spectrophotometry enzyme substrate dilution problem



we wanted to see if different [substrate] affected velocity of rxn with an enzyme

We kept the enzyme volume always 0.2 mL optimal pH is 8. We did everything at pH 7 and total volume in cuvet was 3mL

0.2 mL enzyme + 0.3 mL substrate + 2.5 mL tris = total volume always 3ml
0.2 mL enzyme + 0.9 mL substrated + 1.9 mL tris = total volume always 3ml
Etc..


We would put these cuvets under a spectrophotometer determined %T and graphed it.

Another group did the exact same thing so that we could compare our results to make sure they were similar.

But the group made an error. Instead of a total volume of 3mL they used a total volume of 5 mL

i.e.
0.2 mL enzyme + 0.3 mL substrate + 4.5 mL tris = total volume always 5ml
0.2 mL enzyme + 0.9 mL substrated + 3.9 mL tris = total volume always 5ml
Etc..


They added more Tris. Now their %T values (at 5mL total) are higher than our %T values (at 3ml) so they have a lower [ ] when I used beers law to determine the [ ]. this lower [ ] is really skewing my data.

information i have:
Enzyme = 0.135g/700 mL optimal pH is 8
Tris buffer = 0.05M
Substrate = 1mM

Can I do anything to fix this?
They numbers are really skewing my data. I would prefer not to exclude their data. Any suggestions?



thanks
Reply With Quote
  #2  
Old 02-21-2009, 03:38 PM
NATHANALANPATRICKROGERS's Avatar
Summer Student
Points: 306, Level: 6 Points: 306, Level: 6 Points: 306, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Maroochydore
Posts: 73
Thanks: 4
Thanked 13 Times in 11 Posts
Default Re: spectrophotometry enzyme substrate dilution problem

Use excel to find the equation of the [two] sets of data and solve.
Reply With Quote
  #3  
Old 02-21-2009, 09:34 PM
Pipette Filler
Points: 10, Level: 1 Points: 10, Level: 1 Points: 10, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: spectrophotometry enzyme substrate dilution problem

maybe i'm an idiot but what would i be solving for?


when 0.3 mL substrate was added I graphed the formation of [product ] vs time and calculated the slope which equals initial velocity.

Then for 0.9 mL substrate added I graphed the formation of [product] vs time to get the slope = intial velocity.

etc for each volume of substrate added.

then I graphed initial velocity versus [substrate] to determine the slope.

I did the same thing when the total [ ] was 5mL.



the intial velocity versus [substrate] for volume total 3 mL = 0.0112

the intial velocity versus [substrate] for volume total 5 ml = 0.0054

I don't know how to fix the difference in the numbers.

I was wondering if you could explain what I would be solving for?

I have everything in excel but still not too sure what to do with it.

thanks
Reply With Quote
  #4  
Old 02-23-2009, 12:26 AM
NATHANALANPATRICKROGERS's Avatar
Summer Student
Points: 306, Level: 6 Points: 306, Level: 6 Points: 306, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Maroochydore
Posts: 73
Thanks: 4
Thanked 13 Times in 11 Posts
Default Re: spectrophotometry enzyme substrate dilution problem

There are a number of ways to do this but none are particularly eloquent - unless you have heaps of data and the answer is in fact plausible.

My suspicion is that the substrate -vs- reaction time graph for your enzyme may in fact be a squiggly line [Excel can produce a pretty good polynomial equation to reflect this though].
Reply With Quote
Reply

Tags
dilution , enzyme , problem , spectrophotometry , substrate


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Enzyme Assays peterish Protein Forum 5 05-19-2011 10:56 AM
SDS-PAGE problem Steve Nothwehr Protocols and Methods Forum 2 10-16-2009 08:59 AM
Primer dilution problem in RAPD PCR richadeo Real-Time PCR and Quantitative PCR Forum 6 01-17-2009 09:09 AM
Unidentified problem with low concentrated samples ggerard Western Blot Forum 3 12-22-2008 05:52 PM
Restriction enzyme problem biberj Molecular Cloning Forum 0 12-16-2008 10:29 PM


All times are GMT. The time now is 05:05 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13351 seconds with 16 queries