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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:[log in to unmask]] On Behalf Of Guy Cox
>> Sent: vendredi 8 mars 2013 17:02
>> To: [log in to unmask]
>> 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 [[log in to unmask]]
>> on behalf of Martin Wessendorf [[log in to unmask]]
>> Sent: 09 March 2013 02:31
>> To: [log in to unmask]
>> 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, Micha³ 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: (612) 626-0145
>> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
>> University of Minnesota Preferred FAX: (612) 624-8118
>> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
>> Minneapolis, MN 55455 e-mail: [log in to unmask]
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