10 replies to this topic

[katenkak]

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!

[Doki]

katenkak, on May 20 2009, 03:53 AM, said:

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

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?

[katenkak]

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?

Nabi, on May 20 2009, 05:24 AM, said:

katenkak, on May 20 2009, 03:53 AM, said:

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

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?

[Doki]

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.

[little mouse]

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

[katenkak]

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

Nabi, on May 20 2009, 12:07 PM, said:

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.

[Doki]

katenkak, on May 20 2009, 09:36 PM, said:

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

Nabi, on May 20 2009, 12:07 PM, said:

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!

[katenkak]

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.

little mouse, on May 20 2009, 01:15 PM, said:

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

[little mouse]

katenkak, on May 20 2009, 02:38 PM, said:

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.

little mouse, on May 20 2009, 01:15 PM, said:

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.

[mdfenko]

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).

[katenkak]

Thanks a lot!!!!!

little mouse, on May 20 2009, 03:41 PM, said:

katenkak, on May 20 2009, 02:38 PM, said:

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.

little mouse, on May 20 2009, 01:15 PM, said:

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.