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If preimmune sera and immune sera are both reacting to recombinat antigen in wes


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7 replies to this topic

#1 Inbox

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Posted 05 July 2013 - 11:13 AM

If preimmune sera and immune sera are both reacting to recombinat antigen in western blot. How do you interpret it?
Thanks.

Edited by prabhubct, 05 July 2013 - 11:13 AM.


#2 mdfenko

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Posted 05 July 2013 - 02:57 PM

are they reacting equally?

have you tried just using the secondary antibody?

are you sure the animal was not exposed to the antigen in any way prior to immunization?
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#3 Inbox

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Posted 05 July 2013 - 08:27 PM

immune sera react more strongly, Preimmune sera shows slightly less intensity but sure like positive one(clear Single band).
Yes secondary Ab was used.
SPF animal sera was as Preimmune sera.

#4 Missle

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Posted 08 July 2013 - 05:45 AM

What is the purpose of your western blot? If you are measuring immune response then the differential signal indicates you are mounting an immune response. If you are looking for a specific antibody prep it looks like the SPF serum has antibody against a homologous protein. Are you looking to use the immune serum as serum or purify it?

If you haven't done so already, it would probably be worthwhile to run a blot where you don't use any primary antibody to ensure that the secondary antibody isn't cross-reacting to your protein...

#5 Inbox

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Posted 08 July 2013 - 11:42 AM

Diagnostics western blot.

#6 mdfenko

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Posted 09 July 2013 - 04:08 AM

have you tried just using the secondary antibody?

Yes secondary Ab was used.

what i meant was have you tried using secondary without primary to see if the secondary is reacting with the antigen (as missle recommended).
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#7 Inbox

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Posted 09 July 2013 - 09:04 AM

Oh, Sorry I missed meaning. Will try it.
Isn't Secondary antibody specific for Primary Antibody binding only. e.g. in goat anti-rabbit Ab (Polyclonal right?) must be specific for IgG of Rabbit and should not react to other antigenic portion. or does it include polyclonal Sec. Antibody which in addition to binding to Rabbit IgG, binds to whatever Ag to which goat might have came in contact with?

#8 Missle

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Posted 09 July 2013 - 10:06 AM

The secondary antibody is specific for the primary antibody but there can be cross reactivity. Cross reactivity is more probable when the other protein is an antibody of another species (say the goat anti-rabbit polyclonal produces some cross reactivity with mouse antibody) but it is possible that it can also cross react with a non-IgG protein. I think it is more of a reach but it does happen and so is a reasonable and quick thing to rule out.




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