Hi,
I'm kinda venturing into unknown territory here and I'd like some advice as to which protein tags to chose. I'd like to over-express my gene of interest in zebrafish and because the possibility of getting an antibody specific for my gene is very unlikely I was thinking of attaching a protein tag to the vector construct. My problem is I have no idea what protein tag to use. I know there are differences for His, FLAG, HA and myc tags in terms of using them for downstream protein purification, but for techniques like western-blotting and immunochemistry does it matter which tags I use?
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#2
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Post Dog
Posted 15 September 2010 - 10:47 PM
I found the anti-FLAG antibody M2 (the most common one) is pretty bad for western, much better was the HA antibody I was using. I might have to check which one that was...
rsm
rsm
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#3
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The Goddamn Batman!
Posted 15 September 2010 - 11:18 PM
Rsm, on 15 September 2010 - 10:47 PM, said:
I found the anti-FLAG antibody M2 (the most common one) is pretty bad for western, much better was the HA antibody I was using. I might have to check which one that was...
rsm
rsm
Really? In my hands the M2 from sigma is quite good for WB or Ips! The HA is good as well, but I´d rather have the flag one. You can choose one of those Flag,Ha,Myc, all of them are ok for WB.
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#4
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Posted 16 September 2010 - 01:01 AM
Rsm, on 15 September 2010 - 10:47 PM, said:
I found the anti-FLAG antibody M2 (the most common one) is pretty bad for western, much better was the HA antibody I was using. I might have to check which one that was...
rsm
rsm
It is working fine for me. I have no problem with FLAG. I'm using a kit from Sigma (# FLMC) and it's awesome.
His tag is also famous. But perhaps the most common one is GFP?!
#5
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Posted 16 September 2010 - 01:16 AM
Curtis, on 16 September 2010 - 01:01 AM, said:
Rsm, on 15 September 2010 - 10:47 PM, said:
I found the anti-FLAG antibody M2 (the most common one) is pretty bad for western, much better was the HA antibody I was using. I might have to check which one that was...
rsm
rsm
It is working fine for me. I have no problem with FLAG. I'm using a kit from Sigma (# FLMC) and it's awesome.
His tag is also famous. But perhaps the most common one is GFP?!
yeah but for WB His-tag is not so good, and with GFP you have the problem that the tag is too big, so it is better Flag or Ha. GFP is better for IF. And His is not good for IF
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#6
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Posted 16 September 2010 - 01:43 AM
FLAG-, HA-, myc-, and His-tags are all fine.
GFP is very useful, too, but you should be ware of it when you work with small proteins. GFP is quite big and due to this it may disturb interactions of your protein.
Another important thing is to chose whether you tag your protein N- or C-terminal and whether charges of the tag may interfere (FLAG is negativ, His postive for example).
GFP is very useful, too, but you should be ware of it when you work with small proteins. GFP is quite big and due to this it may disturb interactions of your protein.
Another important thing is to chose whether you tag your protein N- or C-terminal and whether charges of the tag may interfere (FLAG is negativ, His postive for example).
#7
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Posted 16 September 2010 - 01:55 PM
Chakchel, on 16 September 2010 - 01:43 AM, said:
FLAG-, HA-, myc-, and His-tags are all fine.
GFP is very useful, too, but you should be ware of it when you work with small proteins. GFP is quite big and due to this it may disturb interactions of your protein.
Another important thing is to chose whether you tag your protein N- or C-terminal and whether charges of the tag may interfere (FLAG is negativ, His postive for example).
GFP is very useful, too, but you should be ware of it when you work with small proteins. GFP is quite big and due to this it may disturb interactions of your protein.
Another important thing is to chose whether you tag your protein N- or C-terminal and whether charges of the tag may interfere (FLAG is negativ, His postive for example).
Thanks for all the replies. I've got eGFP being produced independently via IRES because I didn't want it to be attached to my protein of interest in case it interferes with protein folding. How do I check whether charges of the tags interfere with my protein? Is there a program I can plop it into to see? I have no experience working with protein so this is all very new to me.
