antibodies for immunofluorescence

[Michał Majkowski]

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Dear All,
I do not know if this is a good place to address my problem as it
regards immunofluorescence. The problem is: we have in the lab
antibodies dedicated to Western Blot (according to manufacturer; Abcam).
We used them to IF and we obtained some nice images. What do you think
about such data? Should it be confirmed by IgG dedicated to IF?   Thank
you for help.
Best,
Michal

[Carol Heckman]

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Michal-
I would think you need to consider whether it is a monoclonal or polyclonal antibody.  If you have a blocking antibody, such as the peptide the antibody is raised against, it would help show that it is a specific identification.  The monoclonal antibody only attaches a single epitope.
Carol Heckman

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Michał Majkowski [[hidden email]]
Sent: Friday, March 08, 2013 3:36 AM
To: [hidden email]
Subject: antibodies for immunofluorescence

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Dear All,
I do not know if this is a good place to address my problem as it
regards immunofluorescence. The problem is: we have in the lab
antibodies dedicated to Western Blot (according to manufacturer; Abcam).
We used them to IF and we obtained some nice images. What do you think
about such data? Should it be confirmed by IgG dedicated to IF?   Thank
you for help.
Best,
Michal

[tineke vendrig]

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It depends on the kind of antibodies. How are these made? In mice ot rats.
or....What is the titer? You can try it anywhere..??
Best,
Tineke Vendrig

2013/3/8 Michał Majkowski <[hidden email]>

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> *****
>
> Dear All,
> I do not know if this is a good place to address my problem as it regards
> immunofluorescence. The problem is: we have in the lab antibodies dedicated
> to Western Blot (according to manufacturer; Abcam). We used them to IF and
> we obtained some nice images. What do you think about such data? Should it
> be confirmed by IgG dedicated to IF?   Thank you for help.
> Best,
> Michal
>

[Johannes Koch]

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Dear Michal,

manufacturers state the standard use which is simply what the antibody
is tested for (and they guarantee that it works). Especially, it is
important to know whether the antibody reacts against the native or the
denatured form of the protein. The latter would be for WB analysis, the
previous for IF. That however, does not exclude that the very same
antibody might also work in the other application. I have had a few
antibodies working in both, some however did not.

As for all images, just do the proper controls, the ones you should also
employ when using an specifically developped antibody, e.g. incubate
with the secondary alone, titrate the antibody, check against your
biological negative control (if you have none, try to come up with one
that makes sense); if you are looking for special subcellular
compartments try to confirm with a second staining (e.g. the ER has
different domains labeled differentially with various antibodies all
sold as if they would recognize "the" ER...) and so on.

So as for your original question: No, I would not do this with other
IgGs - depending on your setup and question I would rather employ other
controls.

Johannes


*Dr. Johannes Koch*

*Tissue Med Biosciences GmbH*

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Am 08.03.2013 15:27, schrieb Tineke Vendrig:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> It depends on the kind of antibodies. How are these made? In mice ot rats.
> or....What is the titer? You can try it anywhere..??
> Best,
> Tineke Vendrig
>
> 2013/3/8 Michał Majkowski <[hidden email]>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> Dear All,
>> I do not know if this is a good place to address my problem as it regards
>> immunofluorescence. The problem is: we have in the lab antibodies dedicated
>> to Western Blot (according to manufacturer; Abcam). We used them to IF and
>> we obtained some nice images. What do you think about such data? Should it
>> be confirmed by IgG dedicated to IF?   Thank you for help.
>> Best,
>> Michal
>>

[Martin Wessendorf-2]

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Dear Michal--

On 3/8/2013 2:36 AM, Michał Majkowski wrote:

> I do not know if this is a good place to address my problem as it
> regards immunofluorescence. The problem is: we have in the lab
> antibodies dedicated to Western Blot (according to manufacturer; Abcam).
> We used them to IF and we obtained some nice images. What do you think
> about such data? Should it be confirmed by IgG dedicated to IF? Thank
> you for help.

The quick and safe answer is that you can never trust any antibody for
any purpose without first characterizing it thoroughly.

Antibodies that work for one application (e.g., westerns) may be
completely unusable for another (e.g., IF)...or they may work perfectly.
  A starting point is to compare the labeling that you obtain, to the
labeling that's previously been published.  The "gold standard" is to
demonstrate that the labeling is not observed in a knockout animal.

Cliff Saper at Journal of Comparative Neurology published an editorial
on antibody use that may be helpful.  It can be found at:
http://www.wiley.com/legacy/wileyblackwell/images/antibody_editorial .

Good luck!

Martin Wessendorf

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Guy Cox-2]

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Martin,

              I must say I am very worried by your statement "Antibodies that work for one application (e.g., westerns) may be completely unusable for another (e.g., IF)".  Can you give me a biological reason for this?  It would seem to make most cell biological work impossible.  I would assume that most of us work the same way as me - ie using microscopy to identify the location, and chromatography to characterise the protein.  If you don't use the same antibody both times you will just be generating nonsense!  

                                                                              Guy

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Martin Wessendorf [[hidden email]]
Sent: 09 March 2013 02:31
To: [hidden email]
Subject: Re: antibodies for immunofluorescence

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Dear Michal--

On 3/8/2013 2:36 AM, Michał Majkowski wrote:

> I do not know if this is a good place to address my problem as it
> regards immunofluorescence. The problem is: we have in the lab
> antibodies dedicated to Western Blot (according to manufacturer; Abcam).
> We used them to IF and we obtained some nice images. What do you think
> about such data? Should it be confirmed by IgG dedicated to IF? Thank
> you for help.

The quick and safe answer is that you can never trust any antibody for
any purpose without first characterizing it thoroughly.

Antibodies that work for one application (e.g., westerns) may be
completely unusable for another (e.g., IF)...or they may work perfectly.
  A starting point is to compare the labeling that you obtain, to the
labeling that's previously been published.  The "gold standard" is to
demonstrate that the labeling is not observed in a knockout animal.

Cliff Saper at Journal of Comparative Neurology published an editorial
on antibody use that may be helpful.  It can be found at:
http://www.wiley.com/legacy/wileyblackwell/images/antibody_editorial .

Good luck!

Martin Wessendorf

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Eric Griffis]

Hi Guy,

I don't think what Martin said is at all controversial.  Some antibodies are very good at recognizing epitopes that are unmasked only after proteins have been denatured by SDS-PAGE, making them useful for western blotting.  Other antibodies can recognize epitopes that are present in the native protein, making them useful for IF, but epitopes can be destroyed by denaturing, which makes antibodies that specifically recognize them bad for westerns.  As long as you have appropriate controls to show the specificity for each antibody in each assay, I see no reason why one couldn't use two different antibodies for the same protein, depending upon the application.

Eric


Eric Griffis
Principal Investigator
Centre for Gene Regulation and Expression
College of Life Sciences
University of Dundee
MSI/WTB/JBC Complex
Dow Street
Dundee DD1 5EH
UK

Tel: [phone]
Fax: [phone]
Email: [hidden email]<mailto:[hidden email]>
Lab Website:  http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html
GRE Website: http://gre.lifesci.dundee.ac.uk



On 8 Mar 2013, at 16:02, Guy Cox wrote:

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Martin,

             I must say I am very worried by your statement "Antibodies that work for one application (e.g., westerns) may be completely unusable for another (e.g., IF)".  Can you give me a biological reason for this?  It would seem to make most cell biological work impossible.  I would assume that most of us work the same way as me - ie using microscopy to identify the location, and chromatography to characterise the protein.  If you don't use the same antibody both times you will just be generating nonsense!

                                                                             Guy

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Martin Wessendorf [[hidden email]]
Sent: 09 March 2013 02:31
To: [hidden email]
Subject: Re: antibodies for immunofluorescence

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Dear Michal--

On 3/8/2013 2:36 AM, Michał Majkowski wrote:

I do not know if this is a good place to address my problem as it
regards immunofluorescence. The problem is: we have in the lab
antibodies dedicated to Western Blot (according to manufacturer; Abcam).
We used them to IF and we obtained some nice images. What do you think
about such data? Should it be confirmed by IgG dedicated to IF? Thank
you for help.

The quick and safe answer is that you can never trust any antibody for
any purpose without first characterizing it thoroughly.

Antibodies that work for one application (e.g., westerns) may be
completely unusable for another (e.g., IF)...or they may work perfectly.
 A starting point is to compare the labeling that you obtain, to the
labeling that's previously been published.  The "gold standard" is to
demonstrate that the labeling is not observed in a knockout animal.

Cliff Saper at Journal of Comparative Neurology published an editorial
on antibody use that may be helpful.  It can be found at:
http://www.wiley.com/legacy/wileyblackwell/images/antibody_editorial .

Good luck!

Martin Wessendorf

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]


The University of Dundee is a registered Scottish Charity, No: SC015096

[Tamara Howard]

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I'll wade in on this one - I tend to do my westerns on samples that have not been fixed (unless I'm trying to do RIP,  my current bugaboo) and that have been denatured, then run on regular old denaturing SDS-PAGE. I prefer to do immunolocalizations (IF, IEM, immunoenzyme for brightfield) on fixed samples, some of which have also been dehydrated and embedded in paraffin or resin. Some antibodies do not recognize their epitopes once those epitopes are fixed, masked, whatever in the supposedly native environment of a cell/tissue BUT they will bind beautifully to a denatured protein on PVDF.

Now I want to work on Guy's samples - life would be much easier if all antibodies worked all of the time, no matter what horrible/necessary things have been done to the sample!

Tamara
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Guy Cox [[hidden email]]
Sent: Friday, March 08, 2013 9:02 AM
To: [hidden email]
Subject: Re: antibodies for immunofluorescence

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Martin,

              I must say I am very worried by your statement "Antibodies that work for one application (e.g., westerns) may be completely unusable for another (e.g., IF)".  Can you give me a biological reason for this?  It would seem to make most cell biological work impossible.  I would assume that most of us work the same way as me - ie using microscopy to identify the location, and chromatography to characterise the protein.  If you don't use the same antibody both times you will just be generating nonsense!

                                                                              Guy

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Martin Wessendorf [[hidden email]]
Sent: 09 March 2013 02:31
To: [hidden email]
Subject: Re: antibodies for immunofluorescence

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Dear Michal--

On 3/8/2013 2:36 AM, Michał Majkowski wrote:

> I do not know if this is a good place to address my problem as it
> regards immunofluorescence. The problem is: we have in the lab
> antibodies dedicated to Western Blot (according to manufacturer; Abcam).
> We used them to IF and we obtained some nice images. What do you think
> about such data? Should it be confirmed by IgG dedicated to IF? Thank
> you for help.

The quick and safe answer is that you can never trust any antibody for
any purpose without first characterizing it thoroughly.

Antibodies that work for one application (e.g., westerns) may be
completely unusable for another (e.g., IF)...or they may work perfectly.
  A starting point is to compare the labeling that you obtain, to the
labeling that's previously been published.  The "gold standard" is to
demonstrate that the labeling is not observed in a knockout animal.

Cliff Saper at Journal of Comparative Neurology published an editorial
on antibody use that may be helpful.  It can be found at:
http://www.wiley.com/legacy/wileyblackwell/images/antibody_editorial .

Good luck!

Martin Wessendorf

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Arne Seitz]

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Guy,

I slightly disagree with you point of view and  I second Martin's opinion.

If you e.g. go to the Millipore website (http://www.millipore.com; no commercial interests) and you search for tubulin antibodies you fill find a variety of products. For each of them you find a key application like e.g. Immunocytochemistry, Immunohistochemistry, Western Blotting or Flow Cytomertry. Some of them are optimized/tested for only one application others can be used for all of them. So there seems to be a difference and it is worth while checking before.

Best regards
Arne

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Guy Cox
> Sent: vendredi 8 mars 2013 17:02
> To: [hidden email]
> Subject: Re: antibodies for immunofluorescence
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Martin,
>
>               I must say I am very worried by your statement "Antibodies that work
> for one application (e.g., westerns) may be completely unusable for another
> (e.g., IF)".  Can you give me a biological reason for this?  It would seem to
> make most cell biological work impossible.  I would assume that most of us
> work the same way as me - ie using microscopy to identify the location, and
> chromatography to characterise the protein.  If you don't use the same
> antibody both times you will just be generating nonsense!
>
>                                                                               Guy
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]]
> on behalf of Martin Wessendorf [[hidden email]]
> Sent: 09 March 2013 02:31
> To: [hidden email]
> Subject: Re: antibodies for immunofluorescence
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Michal--
>
> On 3/8/2013 2:36 AM, Michał Majkowski wrote:
>
> > I do not know if this is a good place to address my problem as it
> > regards immunofluorescence. The problem is: we have in the lab
> > antibodies dedicated to Western Blot (according to manufacturer; Abcam).
> > We used them to IF and we obtained some nice images. What do you think
> > about such data? Should it be confirmed by IgG dedicated to IF? Thank
> > you for help.
>
> The quick and safe answer is that you can never trust any antibody for any
> purpose without first characterizing it thoroughly.
>
> Antibodies that work for one application (e.g., westerns) may be completely
> unusable for another (e.g., IF)...or they may work perfectly.
>   A starting point is to compare the labeling that you obtain, to the labeling
> that's previously been published.  The "gold standard" is to demonstrate that
> the labeling is not observed in a knockout animal.
>
> Cliff Saper at Journal of Comparative Neurology published an editorial on
> antibody use that may be helpful.  It can be found at:
> http://www.wiley.com/legacy/wileyblackwell/images/antibody_editorial .
>
> Good luck!
>
> Martin Wessendorf
>
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]

[Martin Wessendorf-2]

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Dear Guy et al--

On 3/8/2013 10:02 AM, Guy Cox wrote:
>                I must say I am very worried by your statement "Antibodies that work for one application (e.g., westerns) may be completely unusable for another (e.g., IF)".  Can you give me a biological reason for this?  It would seem to make most cell biological work impossible.  I would assume that most of us work the same way as me - ie using microscopy to identify the location, and chromatography to characterise the protein.  If you don't use the same antibody both times you will just be generating nonsense!

The environment in which a protein exists in a western is quite
different from that in which it exists in the cell: in a gel, it hasn't
been fixed (and thus chemically modified) and it may have been subjected
to detergent treatments and other steps that denature it in ways
different from those used in fixed cells or tissue.  For this reason,
the best way to characterize an antibody is to do so using the same
methods in which it is to be used--anything else requires making
assumptions that may or may not be appropriate or accurate.

In the experience of my lab--and I expect in the experience of the
antibody vendors as well, given the fact that they carefully label
antibodies as being useful for one purpose and not another--there
sometimes are antibodies that work for westerns that don't work for
immunofluorescence, and vice-versa.  We also find antibodies that appear
to label a substance specifically in cells or tissue, but that label a
variety of bands in westerns, some of which don't appear to have a clear
relationship to the substance in question.  I think this gets to be more
of a problem when dealing with proteins that aren't very abundant and
when the antibodies are raised against epitopes that are common to a lot
of other proteins.

In any case, my point is not that antibodies that work well for westerns
CAN'T work for IF.  It's that they may not.

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Tim Feinstein-2]

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I sent a private message to the OP, but it may be worth wading into this.  An antibody does not need to have perfect specificity for western blotting, as long as its background bands are of lower affinity and well-separated in size.  People even 'make do' with antibodies that have terribly strong background as long as it keeps its distance from the protein of interest.  The specificity needs for IF are of course much more stringent.  Further, some epitopes are only detectable at an acceptable affinity in the presence of strong detergent denaturation (which may mimic the short peptide that was used as an antigen) or in its absence.  

cheers,


TF

Timothy Feinstein, PhD
Visiting Research Associate
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Mar 8, 2013, at 11:31 AM, Seitz Arne wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Guy,
>
> I slightly disagree with you point of view and  I second Martin's opinion.
>
> If you e.g. go to the Millipore website (http://www.millipore.com; no commercial interests) and you search for tubulin antibodies you fill find a variety of products. For each of them you find a key application like e.g. Immunocytochemistry, Immunohistochemistry, Western Blotting or Flow Cytomertry. Some of them are optimized/tested for only one application others can be used for all of them. So there seems to be a difference and it is worth while checking before.
>
> Best regards
> Arne
>
>
>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Guy Cox
>> Sent: vendredi 8 mars 2013 17:02
>> To: [hidden email]
>> Subject: Re: antibodies for immunofluorescence
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Martin,
>>
>>              I must say I am very worried by your statement "Antibodies that work
>> for one application (e.g., westerns) may be completely unusable for another
>> (e.g., IF)".  Can you give me a biological reason for this?  It would seem to
>> make most cell biological work impossible.  I would assume that most of us
>> work the same way as me - ie using microscopy to identify the location, and
>> chromatography to characterise the protein.  If you don't use the same
>> antibody both times you will just be generating nonsense!
>>
>>                                                                              Guy
>>
>> ________________________________________
>> From: Confocal Microscopy List [[hidden email]]
>> on behalf of Martin Wessendorf [[hidden email]]
>> Sent: 09 March 2013 02:31
>> To: [hidden email]
>> Subject: Re: antibodies for immunofluorescence
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Michal--
>>
>> On 3/8/2013 2:36 AM, Michał Majkowski wrote:
>>
>>> I do not know if this is a good place to address my problem as it
>>> regards immunofluorescence. The problem is: we have in the lab
>>> antibodies dedicated to Western Blot (according to manufacturer; Abcam).
>>> We used them to IF and we obtained some nice images. What do you think
>>> about such data? Should it be confirmed by IgG dedicated to IF? Thank
>>> you for help.
>>
>> The quick and safe answer is that you can never trust any antibody for any
>> purpose without first characterizing it thoroughly.
>>
>> Antibodies that work for one application (e.g., westerns) may be completely
>> unusable for another (e.g., IF)...or they may work perfectly.
>>  A starting point is to compare the labeling that you obtain, to the labeling
>> that's previously been published.  The "gold standard" is to demonstrate that
>> the labeling is not observed in a knockout animal.
>>
>> Cliff Saper at Journal of Comparative Neurology published an editorial on
>> antibody use that may be helpful.  It can be found at:
>> http://www.wiley.com/legacy/wileyblackwell/images/antibody_editorial .
>>
>> Good luck!
>>
>> Martin Wessendorf
>>
>> --
>> Martin Wessendorf, Ph.D.                   office: [phone]
>> Assoc Prof, Dept Neuroscience                 lab: [phone]
>> University of Minnesota             Preferred FAX: [phone]
>> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>> Minneapolis, MN  55455                    e-mail: [hidden email]