Login

Join for Free!
118255 members


Does my protein bind DNA?

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Does my protein bind DNA?

Postby evilid » Wed Nov 26, 2008 6:10 pm

I am working on a nuclear protein with unknown functions.
If I want to test whether this protein has a capacity to bind DNA (or even RNA), what kind of experiments can I do?
Thanks.
evilid
Garter
Garter
 
Posts: 7
Joined: Wed Nov 26, 2008 6:06 pm

Postby mith » Wed Nov 26, 2008 6:21 pm

USE A BEAD!
Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr
User avatar
mith
Inland Taipan
Inland Taipan
 
Posts: 5345
Joined: Thu Jan 20, 2005 8:14 pm
Location: Nashville, TN

Re:

Postby evilid » Wed Nov 26, 2008 9:00 pm

mith wrote:USE A BEAD!

Could you be more specific, please?
evilid
Garter
Garter
 
Posts: 7
Joined: Wed Nov 26, 2008 6:06 pm


Postby mith » Thu Nov 27, 2008 6:05 am

specific? a bead that binds dna of course
Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr
User avatar
mith
Inland Taipan
Inland Taipan
 
Posts: 5345
Joined: Thu Jan 20, 2005 8:14 pm
Location: Nashville, TN

Postby MrMistery » Thu Nov 27, 2008 4:42 pm

there are a number of different experiments you could do to analyze this. Beads are probably the best and the easiest, but you could also do, for example, an electrophoretic mobility shift assay.
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
User avatar
MrMistery
Inland Taipan
Inland Taipan
 
Posts: 6832
Joined: Thu Mar 03, 2005 10:18 pm
Location: Romania(small and unimportant country)

Re:

Postby evilid » Thu Nov 27, 2008 6:08 pm

mith wrote:specific? a bead that binds dna of course


So if my protein is holding a piece of DNA, it will bind beads that has an affinity to DNA.
And use antibodies to detect it on Western maybe?

Do you have any literature I can look up to get more details?
evilid
Garter
Garter
 
Posts: 7
Joined: Wed Nov 26, 2008 6:06 pm

Re:

Postby evilid » Thu Nov 27, 2008 6:11 pm

MrMistery wrote:there are a number of different experiments you could do to analyze this. Beads are probably the best and the easiest, but you could also do, for example, an electrophoretic mobility shift assay.


But you see that I am trying to find whether my protein ever binds to DNA.
Therefore I do not know the target DNA sequence.
I thought I can't do electrophoretic mobility shift assay if I don't know the target sequence.
Right?
evilid
Garter
Garter
 
Posts: 7
Joined: Wed Nov 26, 2008 6:06 pm

Postby Cat » Sun Nov 30, 2008 9:17 pm

Example Protocol:

1. Express protein in question with tag (his tag for example).
2. Purify protein in question and bind it to column through tag (his tag column for example).
3. Isolate total DNA (whatever you want to assay).
4. Add DNA to column containing protein.
5. Wash unbound DNA.
6. Elute protein/DNA mixture - research conditions for binding and washing to make sure not to affect protein-DNA interactions.
7. Proceed with electrophoretic mobility shift assay AND/OR isolate DNA, clone, and sequence.
8. Let us know the outcome.

Good luck!
Cat
King Cobra
King Cobra
 
Posts: 631
Joined: Thu Feb 14, 2008 7:40 pm

Postby mith » Mon Dec 01, 2008 5:34 pm

you can also try putting the protein together with dna, and then denaturing with formaldehyde to make them bind irreversibly then wash away excess dna and then remove formaldehyde. If any dna was bound to the protein, it should come off now.
Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr
User avatar
mith
Inland Taipan
Inland Taipan
 
Posts: 5345
Joined: Thu Jan 20, 2005 8:14 pm
Location: Nashville, TN

Re:

Postby evilid » Mon Dec 01, 2008 10:19 pm

Cat wrote:Example Protocol:

1. Express protein in question with tag (his tag for example).
2. Purify protein in question and bind it to column through tag (his tag column for example).
3. Isolate total DNA (whatever you want to assay).
4. Add DNA to column containing protein.
5. Wash unbound DNA.
6. Elute protein/DNA mixture - research conditions for binding and washing to make sure not to affect protein-DNA interactions.
7. Proceed with electrophoretic mobility shift assay AND/OR isolate DNA, clone, and sequence.
8. Let us know the outcome.

Good luck!


Thank you for the suggestion.
I guess the only tricky part will be the preparation of DNA.
To clone it, I will need manageable sizes of DNA so I might have to treat it with DNAse or nucleases.
How much should I add?

For the electrophoretic mobility shift assay, how would I detect it?
Maybe a Western blot?
evilid
Garter
Garter
 
Posts: 7
Joined: Wed Nov 26, 2008 6:06 pm

Postby Cat » Tue Dec 02, 2008 12:03 am

For DNA prep use kit. Final step should be elution with 50 or 100 ul of water/elution buffer. If you do 100 ul, do it in 2 steps and use 35ul from the 1st elution step for each digestion.

I would suggest to do several (let's say 5-10) 5 and 6 cutter restriction enzyme digestions (done overnight).

For detection you can use either non-denaturing TBE-polyacrylamide gels or TAE-agarose gels - you can also release DNA first and run it on a regular TBE-agarose gel. If sufficient amounts of DNA present, you will be able to visualize with ethidium bromide.

Western is another possibility.
Cat
King Cobra
King Cobra
 
Posts: 631
Joined: Thu Feb 14, 2008 7:40 pm

Re: Does my protein bind DNA?

Postby lsui » Mon May 07, 2012 8:02 pm

A Protein with His-tag may be used in a mobility shift assay?. his-tag may interact inespecifically with DNA?? by electrostatic interactions?.
lsui
Garter
Garter
 
Posts: 1
Joined: Mon May 07, 2012 7:55 pm

Next

Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 2 guests