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
* * * * * 1 votes

Antibody to mouse antigen raised in mice? How it is working?


  • Please log in to reply
10 replies to this topic

#1 katenkak

katenkak

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 52 posts
0
Neutral

Posted 19 May 2009 - 10:53 AM

Hi all! How logical is that: raising monoclonal antibody (which of course will mouse) against mouse protein? I want to use monoclonal antibody against my antigen in mouse neurons, but by all logic I should expect only getting a lot of background, since secondary antibody will be against mouse antigens. Why these antibodies exist at all? Please shed some light on the problem. Thanks all in advance!

#2 Doki

Doki

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 265 posts
2
Neutral

Posted 19 May 2009 - 07:24 PM

Hi all! How logical is that: raising monoclonal antibody (which of course will mouse) against mouse protein? I want to use monoclonal antibody against my antigen in mouse neurons, but by all logic I should expect only getting a lot of background, since secondary antibody will be against mouse antigens. Why these antibodies exist at all? Please shed some light on the problem. Thanks all in advance!

If it exists then there should be a reason for it to be there :D

I am not quite sure what U wanted to ask. U have mouse mAb against mouse proteins? Why would there be a high background then?
Simple living, highnot thinking

#3 katenkak

katenkak

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 52 posts
0
Neutral

Posted 20 May 2009 - 12:30 AM

If primary antibody against mouse protein was raised in mouse, then secondary antibody should anti-mouse. So, when I incubate my fixed mouse neurons or mouse cell extracts, then secondary anti-mouse antibody would react with all mouse immunoglobulins in my mouse cell extracts and not only with primary antibody. Isn't it so?

Hi all! How logical is that: raising monoclonal antibody (which of course will mouse) against mouse protein? I want to use monoclonal antibody against my antigen in mouse neurons, but by all logic I should expect only getting a lot of background, since secondary antibody will be against mouse antigens. Why these antibodies exist at all? Please shed some light on the problem. Thanks all in advance!

If it exists then there should be a reason for it to be there :D

I am not quite sure what U wanted to ask. U have mouse mAb against mouse proteins? Why would there be a high background then?



#4 Doki

Doki

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 265 posts
2
Neutral

Posted 20 May 2009 - 02:07 AM

Ah! I get the picture what U meant. I was a little uncertain of where the problem was.

What is the secondary anti-mouse Ab against? (should be IgG or IgM). Do the cells U r staining express IgG/IgM? If not, they should not be a problem. Otherwise also, you can compare with a control without the primary mAb (the cells + secondary Ab) and see how they compare.
Simple living, highnot thinking

#5 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 20 May 2009 - 03:15 AM

you could also directly label the primary antibody (biotin or Alexa-488)

#6 katenkak

katenkak

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 52 posts
0
Neutral

Posted 20 May 2009 - 04:36 AM

Thank you for reply. I have come to the same conclusion while writing and reading these posts :D

Ah! I get the picture what U meant. I was a little uncertain of where the problem was.

What is the secondary anti-mouse Ab against? (should be IgG or IgM). Do the cells U r staining express IgG/IgM? If not, they should not be a problem. Otherwise also, you can compare with a control without the primary mAb (the cells + secondary Ab) and see how they compare.



#7 Doki

Doki

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 265 posts
2
Neutral

Posted 20 May 2009 - 04:38 AM

Thank you for reply. I have come to the same conclusion while writing and reading these posts :D

Ah! I get the picture what U meant. I was a little uncertain of where the problem was.

What is the secondary anti-mouse Ab against? (should be IgG or IgM). Do the cells U r staining express IgG/IgM? If not, they should not be a problem. Otherwise also, you can compare with a control without the primary mAb (the cells + secondary Ab) and see how they compare.

good luck!
Simple living, highnot thinking

#8 katenkak

katenkak

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 52 posts
0
Neutral

Posted 20 May 2009 - 04:38 AM

Thank you for the suggestion. Do you know very economical procedure for this? I buy commercial antibody, so there are very little and of course expensive-nothing to waste.

you could also directly label the primary antibody (biotin or Alexa-488)



#9 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 20 May 2009 - 05:41 AM

Thank you for the suggestion. Do you know very economical procedure for this? I buy commercial antibody, so there are very little and of course expensive-nothing to waste.

you could also directly label the primary antibody (biotin or Alexa-488)



no economical procedure. It's doubling the price of your antibody if not worse. That's why it would be better to buy it already labeled if possible.
I used reactive biotin from Pierce to label surface proteins (EZ-link sulfo NHs-LC biotin) that worked fine, and for fluorescent labeling, I used a kit from molecular probes to label monoclonal antibodies. But it is expensive (we were producing our own monoclonal antibodies, unless I would have bought them already labeled).
I don't remember exactly the price.

but If you already have the antibody, have a try with and without the primary antibody to see if you really have background. If you are working on tissues, I know there is a treatment to get rid of the Ig inside the tissue, but I don't know how.

#10 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,799 posts
133
Excellent

Posted 20 May 2009 - 05:59 AM

i would expect extra bands rather than a generalized background. these can be discounted by processing the blot without primary antibody (as had been suggested).
talent does what it can
genius does what it must
i do what i get paid to do

#11 katenkak

katenkak

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 52 posts
0
Neutral

Posted 20 May 2009 - 10:49 AM

Thanks a lot!!!!!


Thank you for the suggestion. Do you know very economical procedure for this? I buy commercial antibody, so there are very little and of course expensive-nothing to waste.

you could also directly label the primary antibody (biotin or Alexa-488)



no economical procedure. It's doubling the price of your antibody if not worse. That's why it would be better to buy it already labeled if possible.
I used reactive biotin from Pierce to label surface proteins (EZ-link sulfo NHs-LC biotin) that worked fine, and for fluorescent labeling, I used a kit from molecular probes to label monoclonal antibodies. But it is expensive (we were producing our own monoclonal antibodies, unless I would have bought them already labeled).
I don't remember exactly the price.

but If you already have the antibody, have a try with and without the primary antibody to see if you really have background. If you are working on tissues, I know there is a treatment to get rid of the Ig inside the tissue, but I don't know how.






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

©1999-2013 Protocol Online, All rights reserved.