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Primary antibody too concentrated and diluent incompatibility

IHC antibody western FFPE DAB

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#1 Rnotk

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Posted 25 October 2015 - 10:02 PM

Hi All

 

I have two questions about IHC.

 

1) I recently did IHC staining with concentrated antibody that I purchased, and I prepare three dilutions 1:50, 1:100, 1:200 (Company recommendation was 1:100).

The result was interesting that 1:100 was the best staining, but 1:50 and 1:200 came out as WEAK staining.

 

It is understandable that 1:200 came out weaker than 1:100, but why 1:50 could be weaker than 1:100???? (there was no background).

I repeated experiment (to make sure this is not due to human error), but the result was the same.

 

Are there any explanation of too high concentration of primary antibody affect the IHC efficiency????

By the way, the sample was FFPE human tissue and detection system is polymer HRP with DAB detection.

If there is any similar example in even different conditions (frozen section, ABC detection, fluorescent detection, or even Western blot etc), please let me know. Those might gives me some idea.

 

2) This is different incident, but I realized there is incompatibility of antibody diluent. That means if I use certain antibody with certain diluent, the staining did not come out. Both antibody and diluent were purchased. So I am not sure what exactly are in those, but I am very curious.

I have several diluent solutions and those are sold as universal diluent for antibody. So I am fairly surprised there is incompatibility.

Do you guys have any idea what in those diluent could be creating the incompatibility???

My first guess was sodium azide concentration, but I feel there is more to it.

 

Let me know any information or ideas for either question or both.

 

Thank you guys!!!!

 



#2 mdfenko

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Posted 26 October 2015 - 04:20 AM

1 may be due to steric hindrance?

 

2 may be due to non-specific (or specific) binding to the blocking agent in the diluent. to what is the disappearing primary specific?


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#3 Rnotk

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Posted 26 October 2015 - 02:46 PM

Hi Thanks for the quick reply,

 

For 1,

I think I heard about this interaction, but I was not really sure that could happen in such simple IHC with 1:50 dilution.

I had antibody before which I can only dilute up to 1:10, and never observed something like this time.

 

2. 

I agree, but I just dont know what could be?, The antibody is against human oncogenic protein and should present cytoplasm. The diluent was sold as universal diluent, so it should be able to be used for any antibodies I assume. I successfully stained with this antibody if I use different diluent, which I purchased from different company.

I am just not releasing the name of company for antibody, or both diluent to not to create too much problem as I have no idea what is the source of this issue.

Let me know what ingredient in diluent could be creating binding.

 

I though the diluent is typically PBS based with Na Az for preservative, some protein block (maybe BSA or serum, casein) and some detergent?

As far as I think of none of these should hinder the antibody reactivity.....right??????



#4 mdfenko

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Posted 27 October 2015 - 04:12 AM

1- dilutions are based on antibody titer. your 1:10 antibody was very low titer. this one is higher.

 

with more primary antibody bound close together, may hinder binding of secondary antibody.

 

2- "universal" diluent may not be so universal. for instance, if the blocking agent is casein then you may not be able to use it for many phosphorylation specific antibodies.

 

there may be pH, salt, additive differences which may affect binding or activity of linked enzyme.

 

maybe this universal diluent is good for fluorescent tags or phosphatase based detection systems?


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#5 Rnotk

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Posted 27 October 2015 - 10:00 AM

Thanks again mdfenko.

 

For 1, I was not paying attention to the binding of 2ndary. Thats a good point.

 

For 2, This diluent is known as universal and sold by fairly well known company.

In fact, many antibody works well with this diluent and many research labs and hospitals are using this for routine IHC staining.  

So it is somewhat surprising when I found that there is an antibody incompatible to this diluent.

 

But Thanks for the reply, your reply gave me good perspective.



#6 mdfenko

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Posted 28 October 2015 - 03:40 AM

just a small addendum, when using immunoaffinity chromatography, most times low pH is required to elute the protein from the antibody. once in a while we would use an antibody which requires high pH to elute. you may have a similar situation.


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