Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

tagged protein size


  • Please log in to reply
10 replies to this topic

#1 zogene

zogene

    member

  • Active Members
  • Pip
  • 24 posts
3
Neutral

Posted 30 January 2013 - 02:33 PM

Hello

I am wondering about western blotting for tagged protein with different Abs, if I have protein X with any kind of tag lets say His the expected size will be His+X and when I satin it with His Ab, what if I have X Ab does this protein give me the same size or just X size?

many thanks

"Be the change that you wish to see in the world"


#2 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,113 posts
59
Excellent

Posted 30 January 2013 - 06:51 PM

x+His size. His is very small.

#3 zogene

zogene

    member

  • Active Members
  • Pip
  • 24 posts
3
Neutral

Posted 30 January 2013 - 07:31 PM

thanks for replying, no His is not small it is around 15-20 kd I think
regards

"Be the change that you wish to see in the world"


#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,505 posts
253
Excellent

Posted 30 January 2013 - 07:53 PM

A typical His tag is 6 His amino acids at either the C or N terminus. This will have a MW of around 155*6 = 930 larger than the original protein.

#5 zogene

zogene

    member

  • Active Members
  • Pip
  • 24 posts
3
Neutral

Posted 30 January 2013 - 08:00 PM

hi
I did not understand very well lets say I have pEt vector with his tagged when I induce it alone with IPTG I have about 15-20 kd band so I suppose when I induce my recobinant protein will be x +20, or I did not understand very well?
many thanks I am just new with proteins

"Be the change that you wish to see in the world"


#6 zogene

zogene

    member

  • Active Members
  • Pip
  • 24 posts
3
Neutral

Posted 30 January 2013 - 08:06 PM

by the way can any one put tagged common protein size like c-Myc/ His/GST/S/Flag/HA
and is tagged usually very small or it can be big size? finally sometimes it mentioned this plamid tagged with X which express protein with X kd? how the tagged express as big but the real size is very small?
many thanks

"Be the change that you wish to see in the world"


#7 neuron

neuron

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 187 posts
5
Neutral

Posted 30 January 2013 - 08:50 PM

@Zogene, I don't know if I have understood your last comment correctly. But I think you are confused with empty plasmid size. Like Phage said His mol wt is 155 and if your protein size is 40 kda , the recombinant protein with His tag will be 40,000+930=40930 da. which is just increase of ~1kda . The pET vector that you are inducing alone does not contain your gene, that is why it gives you 15-20 kda (~136-180 amino acids). you need to calculate from the vector sequence what are these amino acids. I hope I am making sensePosted Image , please somebody correct me if I am wrong.

#8 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,113 posts
59
Excellent

Posted 30 January 2013 - 10:49 PM

lol, I work on L protein of NDV virus which is 249 kD. For me 15 kD is small.

#9 zogene

zogene

    member

  • Active Members
  • Pip
  • 24 posts
3
Neutral

Posted 30 January 2013 - 11:39 PM

Hi
thanks again for response, ok I got the whole point but in many cases I notice that my protein size is bigger than it should be like in X protein size around 20 kd with His which I supposed it should be 20 either so my protin appeared around 40 kd I checked the frame and there nothing with it, that is what I could not explaine it?

many thanks

"Be the change that you wish to see in the world"


#10 neuron

neuron

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 187 posts
5
Neutral

Posted 31 January 2013 - 12:14 AM

This is a very common problem, you should check the other thread also-

http://www.protocol-...erent-mobility/

#11 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,113 posts
59
Excellent

Posted 31 January 2013 - 02:06 AM

Hi
thanks again for response, ok I got the whole point but in many cases I notice that my protein size is bigger than it should be like in X protein size around 20 kd with His which I supposed it should be 20 either so my protin appeared around 40 kd I checked the frame and there nothing with it, that is what I could not explaine it?

many thanks


Did you read Phage434's reply at all?




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.