[Histonet] RE: protein concentration on tissue culture supernatant

[Thurby, Christina]

Hi Katy,
I have a biotinylation kit from Sigma.  I need to know the starting concentration to run the biotinylation assay.  I ran a Bradford on the antibody yesterday and got a total protein concentration of 10 mg/mL.  Not sure how much of that total protein is my target antibody.  I don't have to purify the antibody, but I do have to dialyze the antibody to remove the sodium azide.  Yes, I am using this antibody on Mouse frozen sections tissue (but the primary is a rabbit monoclonal - so mouse on mouse is not an issue).  I do need to biotinylate the primary antibody to use it with the IHC for LCM kit from molecular devices.  I have tried adding in the biotinylated secondary step (although this is not suggested by the manufacturer) and I'm not getting good results yet.  I have not had any luck finding a commercially available biotinylated calcitonin antibody.  We'll see, I'm still working through this.  Thanks for the feedback.

I'll also include the feedback I rec'd from Dr. van der Loos.  Anyone with more thoughts, please feel free to join the discussion!!!  Many thanks


Hi Christina,
If it is a hybridoma antibody it will contain fetal calf serum for sure.
Again I find it almost unthinkable that we pay 400-700 Euro's/USD's/whatever (or more!) for an antibody that is not characterized concerning its Ig concentration! We had the same problem with ThermoFisher concerning CD3, SP7 when we wanted to perform a conc. matched rabbit negative control.

NB: The ARKit will not work for your rabbit mononclonals. You better turn to the Invitrogen Zenon kit. I do hope these idiots have finally inserted a data sheet and working manual with those kits.... :-(

There is still a lot to improve out there!

Good luck,
Chris


________________________________________
Van: Thurby, Christina [christina.thurby <@t> bms.com]
Verzonden: vrijdag 12 oktober 2012 16:08
Aan: C.M. van der Loos; JMyers1 <@t> aol.com; ihcrg <@t> googlegroups.com
CC: Simutis, Frank
Onderwerp: RE: [IHCRG] IHC question/help
Thanks Chris,
I was very concerned about whether I should be confident with the Bradford results for total protein.  I ran it in triplicate yesterday and my antibody protein concentration measurement is 10 mg/mL (I don't know if there is carrier protein - I will call the vendor again).  The spec sheet lists under Format:  This antibody is supplied as a tissue culture supernatant containing sodium azide as a preservative.  I called the vendor again yesterday to get more information, this is a rabbit monoclonal calcitonin antibody, clone SP17 from a hybridoma.  The vendor reports that it is not purified.  I have to admit, I do not have a good understanding of rabbit monoclonal antibodies just yet (but that is a separate issue all together).

I am using a new test system for a florescent IHC staining kit for LCM from molecular devices.  This kit is for frozen tissue and I need to biotinylate the primary (since this is a direct label kit) and part of the focus is RNA degradation of the sample.  This kit tells me I need to label the samples with 30-100ug/mL of the primary antibody.  This is a much, much higher concentration than I am used to working with.  I have successfully used the ARK kit with other antibodies and I agree - with your comments!   That may be an option we switch back to.
Thanks again for your feedback!

Christina Thurby
Bristol Myers Squibb
812-307-2093



From: C.M. van der Loos [mailto:c.m.vanderloos <@t> amc.uva.nl]
Sent: Friday, October 12, 2012 8:13 AM
To: JMyers1 <@t> aol.com; ihcrg <@t> googlegroups.com
Cc: Thurby, Christina
Subject: RE: [IHCRG] IHC question/help

Dear Christina,
This is way off a dumb question! It's a dumb thing it is a true problem to you and many others. Companies should just mention the specific Ig concentration in a data sheet!
Running a Bradford also picks up the carrier protein (BSA, gelatin, etc) and doesn't help you out. The holds true for a true biotinylation or conjugation reaction: the carrier protein will be labeled as well. You may try the Dako ARKit or Invitrogen Zenon kit. These work with biotinylated anti-mouse Ig Fab-fragments and therefore the carrier protein is not disturbing. Problem again is that you have to know the specific mouse Ig concentration for optimal labeling. One trick helped us out a couple of times:
Optimal IHC is usually performed at 0.25 - 10 ug/ml antibody concentration. So if your unlabeled antibody is staining well you may anticipate the Ig concentration is in that range. Based on the optimal dilution of your unlabeled antibody set up ARK or Zenon labeling assuming the starting Ig concentration is 0.25, 1.50 or 10 ug/ml and then see what comes out best.

Good luck!
Cheers, Chris

Chris van der Loos, PhD
Academic Medical Center
Dept. of Pathology
Amsterdam
The Netherlands

Kristie



>-----Original Message-----
>From: Milne, Katy [mailto:kmilne <@t> bccancer.bc.ca]
>Sent: Friday, October 12, 2012 2:30 PM
>To: histonet <@t> lists.utsouthwestern.edu; Thurby, Christina
>Subject: RE: protein concentration on tissue culture supernatant
>
>Hey Christina,
>
>I'm not sure how much the Bradford will help as there will likely be a lot of other
>proteins in the medium that the antibody is in (ie FBS etc) as you mention.  You
>wouldn't just get the Ab concentration.  Maybe for some biotinylation kits this isn't a
>problem but I'm not sure what you're using.
>
>Do you have a kit that you can straight up biotinylate the Ab while it's still in the
>medium or do you need to purify it?  Does it need to be biotinylated or can you come in
>with a biotinylated secondary (or are you dealing with mouse-on-mouse problems)?
>
>Katy
>
>Message: 3
>Date: Thu, 11 Oct 2012 15:05:39 -0400
>From: "Thurby, Christina" <christina.thurby <@t> bms.com>
>Subject: [Histonet] protein concentration on tissue culture
>       supernatant
>To: "histonet <@t> lists.utsouthwestern.edu"
>       <histonet <@t> lists.utsouthwestern.edu>
>Message-ID:
>       <E9C77491626EBF41BD449EA1098B04060871A45AA7 <@t> ushpwbmsmmp0
>07.one.ads.bms.com>
>
>Content-Type: text/plain; charset="us-ascii"
>
>Please don't blast me if this is a really dumb question.  I have a tissue culture
>supernatant antibody.  I need to know the protein concentration - called the vendor, it is
>unknown.  Does anyone know if I can just run a Bradford to get the protein
>concentration - or will it be irrelevant do to other proteins present in the antibody?  This
>is an IVD antibody, so I expect it to be specific for the target antigen.  This is an
>unconjugated antibody and I want to biotinylate it, but I need a starting protein
>concentration.  Anyone out there with experience doing this with a tissue culture
>supernatant antibody?
>
>Christina Thurby
>Bristol Myers Squibb
>812-307-2093
>


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