(no subject)
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Hi List, One of my colleagues is trying to visualize a nuclear
protein using primary antibodies. Is there a special trick or protocol that
allows the primary and the fluorescent secondary antibodies better access to proteins
inside the nucleus? They have already considered using either Triton or Tween-20. Thanks, Neeraj. Neeraj V. Gohad, Ph.D. Postdoctoral Fellow Okeanos Research Group Department of Biological Sciences 132 Long Hall Clemson University Clemson,SC-29634 Phone: 864-656-3597 Fax: 864-656-0435 Please note my new email address: [hidden email] |
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Neeraj,
You don't mention the fixation protocol used. If and when to use
Triton (0.2-0.5%; harsh) or Tween (gentler) in part depends on the
fixation protocol. Staining nuclear proteins presents its own problems
aside from simply permeabilizing the membrane barriers that let your
antibodies in. Non-specific labeling is often greatly enhanced because
of local ionic strength, pH and very strong electrostatic
interactions. You could have a very high affinity antibody (primary
and/or secondary) and still end up with diffuse (as in worthless)
staining.
Cold methanol/acetone fixation might be the way to go if PFA
(2-3%) plus Triton (0.5%) doesn't seem to work. Also, staining using
higher ionic strength can be helpful and consider conjugating a
fluorophore to your primary and forget the secondary altogether. How
you block your cells can also be very important.
Mario
Hi List, One of my colleagues is trying to visualize a nuclear protein using primary antibodies. Is there a special trick or protocol that allows the primary and the fluorescent secondary antibodies better access to proteins inside the nucleus? They have already considered using either Triton or Tween-20. Thanks, Neeraj. Neeraj V. Gohad, Ph.D. Postdoctoral Fellow Okeanos Research Group Department of Biological Sciences 132 Long Hall Clemson University Clemson,SC-29634 Phone: 864-656-3597 Fax: 864-656-0435 Please note my new email address: [hidden email] -- ________________________________________________________________________________
Mario M. Moronne, Ph.D. [hidden email] [hidden email] |
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In reply to this post by Neeraj Gohad-2
I used to have pretty good success accessing DNA binding
proteins using 5% triton for about 15minutes.
Cold MetOH fixation also worked well...
Good luck!
d
Danielle
Crippen Morphology
and Imaging Core Manager Buck
Institute for Age Research
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Neeraj Gohad Sent: Thursday, January 28, 2010 10:05 AM To: [hidden email] Subject: Hi List, One of my colleagues is trying to visualize a nuclear protein
using primary antibodies. Is there a special trick or protocol that allows
the primary and the fluorescent secondary antibodies better access to proteins
inside the nucleus? They have already considered using either Triton or
Tween-20. Thanks, Neeraj. Neeraj V. Gohad,
Ph.D. Postdoctoral
Fellow Okeanos Research
Group Department of
Biological Sciences 132 Long
Hall Clemson
University Clemson,SC-29634 Phone:
864-656-3597 Fax:
864-656-0435 Please note my
new email address: [hidden email] |
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Woops...meant 0.5% triton...
:)d
Danielle Crippen Morphology
and Imaging Core From: Danielle Crippen Sent: Thursday, January 28, 2010 10:48 AM To: 'Confocal Microscopy List' Subject: RE: I used to have pretty good success accessing DNA binding
proteins using 5% triton for about 15minutes.
Cold MetOH fixation also worked well...
Good luck!
d
Danielle
Crippen Morphology
and Imaging Core Manager Buck
Institute for Age Research
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Neeraj Gohad Sent: Thursday, January 28, 2010 10:05 AM To: [hidden email] Subject: Hi List, One of my colleagues is trying to visualize a nuclear protein
using primary antibodies. Is there a special trick or protocol that allows
the primary and the fluorescent secondary antibodies better access to proteins
inside the nucleus? They have already considered using either Triton or
Tween-20. Thanks, Neeraj. Neeraj V. Gohad,
Ph.D. Postdoctoral
Fellow Okeanos Research
Group Department of
Biological Sciences 132 Long
Hall Clemson
University Clemson,SC-29634 Phone:
864-656-3597 Fax:
864-656-0435 Please note my
new email address: [hidden email] |
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|
In reply to this post by Mario-2
Hi Mario, This is a new project for my colleague, they have tried using a
standard fixation protocol (4% buffered PFA, 0.5% Triton, block with BSA etc..)
to no avail. Best, Neeraj. Neeraj V.
Gohad, Ph.D. Postdoctoral
Fellow Okeanos
Research Group Department of
Biological Sciences 132 Long Hall Clemson
University Clemson,SC-29634 Phone:
864-656-3597 Fax:
864-656-0435 Please note
my new email address: [hidden email] From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Mario Neeraj, You don't mention the fixation protocol used. If and when to
use Triton (0.2-0.5%; harsh) or Tween (gentler) in part depends on the fixation
protocol. Staining nuclear proteins presents its own problems aside from simply
permeabilizing the membrane barriers that let your antibodies in. Non-specific
labeling is often greatly enhanced because of local ionic strength, pH and very
strong electrostatic interactions. You could have a very high affinity antibody
(primary and/or secondary) and still end up with diffuse (as in worthless)
staining. Cold methanol/acetone fixation might be the way to go if PFA
(2-3%) plus Triton (0.5%) doesn't seem to work. Also, staining using higher
ionic strength can be helpful and consider conjugating a fluorophore to your
primary and forget the secondary altogether. How you block your cells can also
be very important. Mario
-- ________________________________________________________________________________ |
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I may have missed a post - what kind of samples are these?
Cultured cells, whole mount tissue/embryos, sections (cryo/paraffin/resin)? Tamara On Thu, 28 Jan 2010 15:09:42 -0500 Neeraj Gohad <[hidden email]> wrote: > Hi Mario, > > This is a new project for my colleague, they have tried >using a standard fixation protocol (4% buffered PFA, 0.5% >Triton, block with BSA etc..) to no avail. > > > Best, > > Neeraj. > > Neeraj V. Gohad, Ph.D. > Postdoctoral Fellow > Okeanos Research Group > Department of Biological Sciences > 132 Long Hall > Clemson University > Clemson,SC-29634 > Phone: 864-656-3597 >Fax: 864-656-0435 > > Please note my new email address: >[hidden email]<mailto:[hidden email]> > > >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of >Mario > Sent: Thursday, January 28, 2010 1:44 PM > To: [hidden email] > Subject: staining nuclear protein > > Neeraj, > > You don't mention the fixation protocol used. If and >when to use Triton (0.2-0.5%; harsh) or Tween (gentler) >in part depends on the fixation protocol. Staining >nuclear proteins presents its own problems aside from >simply permeabilizing the membrane barriers that let your >antibodies in. Non-specific labeling is often greatly >enhanced because of local ionic strength, pH and very >strong electrostatic interactions. You could have a very >high affinity antibody (primary and/or secondary) and >still end up with diffuse (as in worthless) staining. > > Cold methanol/acetone fixation might be the way to go if >PFA (2-3%) plus Triton (0.5%) doesn't seem to work. Also, >staining using higher ionic strength can be helpful and >consider conjugating a fluorophore to your primary and >forget the secondary altogether. How you block your cells >can also be very important. > > Mario > > > Hi List, > > One of my colleagues is trying to visualize a nuclear >protein using primary antibodies. Is there a special >trick or protocol that allows the primary and the >fluorescent secondary antibodies better access to >proteins inside the nucleus? They have already considered >using either Triton or Tween-20. > > Thanks, > > Neeraj. > > Neeraj V. Gohad, Ph.D. > Postdoctoral Fellow > Okeanos Research Group > Department of Biological Sciences > 132 Long Hall > Clemson University > Clemson,SC-29634 > Phone: 864-656-3597 >Fax: 864-656-0435 > > Please note my new email address: >[hidden email]<mailto:[hidden email]> > > > > > > -- > ________________________________________________________________________________ > Mario M. Moronne, Ph.D. > > [hidden email] > [hidden email] *************************** Tamara Howard Cell Biology & Physiology UNM-HSC Albuquerque, NM *************************** |
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Hi Tamara,
I apologize for not including details, my colleague is using cultured neurobalstoma cells grown as a monolayer on coverslips. Neeraj. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tamara A Howard Sent: Thursday, January 28, 2010 3:13 PM To: [hidden email] Subject: Re: staining nuclear protein I may have missed a post - what kind of samples are these? Cultured cells, whole mount tissue/embryos, sections (cryo/paraffin/resin)? Tamara On Thu, 28 Jan 2010 15:09:42 -0500 Neeraj Gohad <[hidden email]> wrote: > Hi Mario, > > This is a new project for my colleague, they have tried >using a standard fixation protocol (4% buffered PFA, 0.5% >Triton, block with BSA etc..) to no avail. > > > Best, > > Neeraj. > > Neeraj V. Gohad, Ph.D. > Postdoctoral Fellow > Okeanos Research Group > Department of Biological Sciences > 132 Long Hall > Clemson University > Clemson,SC-29634 > Phone: 864-656-3597 >Fax: 864-656-0435 > > Please note my new email address: >[hidden email]<mailto:[hidden email]> > > >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of >Mario > Sent: Thursday, January 28, 2010 1:44 PM > To: [hidden email] > Subject: staining nuclear protein > > Neeraj, > > You don't mention the fixation protocol used. If and >when to use Triton (0.2-0.5%; harsh) or Tween (gentler) >in part depends on the fixation protocol. Staining >nuclear proteins presents its own problems aside from >simply permeabilizing the membrane barriers that let your >antibodies in. Non-specific labeling is often greatly >enhanced because of local ionic strength, pH and very >strong electrostatic interactions. You could have a very >high affinity antibody (primary and/or secondary) and >still end up with diffuse (as in worthless) staining. > > Cold methanol/acetone fixation might be the way to go if >PFA (2-3%) plus Triton (0.5%) doesn't seem to work. Also, >staining using higher ionic strength can be helpful and >consider conjugating a fluorophore to your primary and >forget the secondary altogether. How you block your cells >can also be very important. > > Mario > > > Hi List, > > One of my colleagues is trying to visualize a nuclear >protein using primary antibodies. Is there a special >trick or protocol that allows the primary and the >fluorescent secondary antibodies better access to >proteins inside the nucleus? They have already considered >using either Triton or Tween-20. > > Thanks, > > Neeraj. > > Neeraj V. Gohad, Ph.D. > Postdoctoral Fellow > Okeanos Research Group > Department of Biological Sciences > 132 Long Hall > Clemson University > Clemson,SC-29634 > Phone: 864-656-3597 >Fax: 864-656-0435 > > Please note my new email address: >[hidden email]<mailto:[hidden email]> > > > > > > -- > ________________________________________________________________________________ > Mario M. Moronne, Ph.D. > > [hidden email] > [hidden email] *************************** Tamara Howard Cell Biology & Physiology UNM-HSC Albuquerque, NM *************************** |
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Neeraj,
Can you define failure? No staining, gross non-specific staining, lack of expected labeling morphology? If you don't get any signal then the antibodies are probably not working. Try new antibodies. In the nucleus the antibody might be unable to "find" its target epitope. It might be comprised through binding to the high concentrations of nucleic acid, matrix proteins, etc. Labeling at elevated temperature might help (37°C) or playing with the pH and elevated ionic strength or staining for a long time (overnight in the refrigerator). But it does depend on what you mean by failure. BTW, I have never found BSA to be a very good blocker, but that is a specificity issue not an invisibility one. Stickiness inside the nucleus translates into a reduced rate of diffusion. To a first order, the diffusion constant of a large protein will decrease in proportion to the apparent partitioning coefficient of the protein to the sticky environment it interacts with and depends on the antibody's charge, etc. So something that would normally label in 15 minutes could take a few hours at rm. temperature. Mario >Hi Tamara, > >I apologize for not including details, my >colleague is using cultured neurobalstoma cells >grown as a monolayer on coverslips. > > >Neeraj. > > > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On >Behalf Of Tamara A Howard >Sent: Thursday, January 28, 2010 3:13 PM >To: [hidden email] >Subject: Re: staining nuclear protein > >I may have missed a post - what kind of samples are these? >Cultured cells, whole mount tissue/embryos, sections >(cryo/paraffin/resin)? > >Tamara > >On Thu, 28 Jan 2010 15:09:42 -0500 > Neeraj Gohad <[hidden email]> wrote: >> Hi Mario, >> >> This is a new project for my colleague, they have tried >>using a standard fixation protocol (4% buffered PFA, 0.5% >>Triton, block with BSA etc..) to no avail. >> >> >> Best, >> >> Neeraj. >> >> Neeraj V. Gohad, Ph.D. >> Postdoctoral Fellow >> Okeanos Research Group >> Department of Biological Sciences >> 132 Long Hall >> Clemson University >> Clemson,SC-29634 >> Phone: 864-656-3597 >>Fax: 864-656-0435 >> >> Please note my new email address: >>[hidden email]<mailto:[hidden email]> >> >> >>From: Confocal Microscopy List >>[mailto:[hidden email]] On Behalf Of >>Mario >> Sent: Thursday, January 28, 2010 1:44 PM >> To: [hidden email] >> Subject: staining nuclear protein >> >> Neeraj, >> >> You don't mention the fixation protocol used. If and >>when to use Triton (0.2-0.5%; harsh) or Tween (gentler) >>in part depends on the fixation protocol. Staining >>nuclear proteins presents its own problems aside from >>simply permeabilizing the membrane barriers that let your >>antibodies in. Non-specific labeling is often greatly >>enhanced because of local ionic strength, pH and very >>strong electrostatic interactions. You could have a very >>high affinity antibody (primary and/or secondary) and >>still end up with diffuse (as in worthless) staining. >> >> Cold methanol/acetone fixation might be the way to go if >>PFA (2-3%) plus Triton (0.5%) doesn't seem to work. Also, >>staining using higher ionic strength can be helpful and >>consider conjugating a fluorophore to your primary and >>forget the secondary altogether. How you block your cells >>can also be very important. >> >> Mario >> >> >> Hi List, >> >> One of my colleagues is trying to visualize a nuclear >>protein using primary antibodies. Is there a special >>trick or protocol that allows the primary and the >>fluorescent secondary antibodies better access to >>proteins inside the nucleus? They have already considered >>using either Triton or Tween-20. >> >> Thanks, >> >> Neeraj. >> >> Neeraj V. Gohad, Ph.D. >> Postdoctoral Fellow >> Okeanos Research Group >> Department of Biological Sciences >> 132 Long Hall >> Clemson University >> Clemson,SC-29634 >> Phone: 864-656-3597 >>Fax: 864-656-0435 >> >> Please note my new email address: > >[hidden email]<mailto:[hidden email]> >> >> >> >> >> >> -- >> >>________________________________________________________________________________ >> Mario M. Moronne, Ph.D. >> >> [hidden email] >> [hidden email] > >*************************** >Tamara Howard >Cell Biology & Physiology >UNM-HSC >Albuquerque, NM >*************************** -- ________________________________________________________________________________ Mario M. Moronne, Ph.D. [hidden email] [hidden email] |
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Thanks Mario, I think the problem they were having was of access as you are pointing out. They tested the antibodies against western blots of nuclear extract where it showed good specificity and a good signal. When they tried doing IF (which is only a couple of times) they got inconsistent staining and weak signal. I am advising them to do series of labeling experiments at different temperatures using variety of fixatives, fixation times and permebalizing agents.
Neeraj. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mario Sent: Thursday, January 28, 2010 5:09 PM To: [hidden email] Subject: Re: staining nuclear protein Neeraj, Can you define failure? No staining, gross non-specific staining, lack of expected labeling morphology? If you don't get any signal then the antibodies are probably not working. Try new antibodies. In the nucleus the antibody might be unable to "find" its target epitope. It might be comprised through binding to the high concentrations of nucleic acid, matrix proteins, etc. Labeling at elevated temperature might help (37°C) or playing with the pH and elevated ionic strength or staining for a long time (overnight in the refrigerator). But it does depend on what you mean by failure. BTW, I have never found BSA to be a very good blocker, but that is a specificity issue not an invisibility one. Stickiness inside the nucleus translates into a reduced rate of diffusion. To a first order, the diffusion constant of a large protein will decrease in proportion to the apparent partitioning coefficient of the protein to the sticky environment it interacts with and depends on the antibody's charge, etc. So something that would normally label in 15 minutes could take a few hours at rm. temperature. Mario >Hi Tamara, > >I apologize for not including details, my >colleague is using cultured neurobalstoma cells >grown as a monolayer on coverslips. > > >Neeraj. > > > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On >Behalf Of Tamara A Howard >Sent: Thursday, January 28, 2010 3:13 PM >To: [hidden email] >Subject: Re: staining nuclear protein > >I may have missed a post - what kind of samples are these? >Cultured cells, whole mount tissue/embryos, sections >(cryo/paraffin/resin)? > >Tamara > >On Thu, 28 Jan 2010 15:09:42 -0500 > Neeraj Gohad <[hidden email]> wrote: >> Hi Mario, >> >> This is a new project for my colleague, they have tried >>using a standard fixation protocol (4% buffered PFA, 0.5% >>Triton, block with BSA etc..) to no avail. >> >> >> Best, >> >> Neeraj. >> >> Neeraj V. Gohad, Ph.D. >> Postdoctoral Fellow >> Okeanos Research Group >> Department of Biological Sciences >> 132 Long Hall >> Clemson University >> Clemson,SC-29634 >> Phone: 864-656-3597 >>Fax: 864-656-0435 >> >> Please note my new email address: >>[hidden email]<mailto:[hidden email]> >> >> >>From: Confocal Microscopy List >>[mailto:[hidden email]] On Behalf Of >>Mario >> Sent: Thursday, January 28, 2010 1:44 PM >> To: [hidden email] >> Subject: staining nuclear protein >> >> Neeraj, >> >> You don't mention the fixation protocol used. If and >>when to use Triton (0.2-0.5%; harsh) or Tween (gentler) >>in part depends on the fixation protocol. Staining >>nuclear proteins presents its own problems aside from >>simply permeabilizing the membrane barriers that let your >>antibodies in. Non-specific labeling is often greatly >>enhanced because of local ionic strength, pH and very >>strong electrostatic interactions. You could have a very >>high affinity antibody (primary and/or secondary) and >>still end up with diffuse (as in worthless) staining. >> >> Cold methanol/acetone fixation might be the way to go if >>PFA (2-3%) plus Triton (0.5%) doesn't seem to work. Also, >>staining using higher ionic strength can be helpful and >>consider conjugating a fluorophore to your primary and >>forget the secondary altogether. How you block your cells >>can also be very important. >> >> Mario >> >> >> Hi List, >> >> One of my colleagues is trying to visualize a nuclear >>protein using primary antibodies. Is there a special >>trick or protocol that allows the primary and the >>fluorescent secondary antibodies better access to >>proteins inside the nucleus? They have already considered >>using either Triton or Tween-20. >> >> Thanks, >> >> Neeraj. >> >> Neeraj V. Gohad, Ph.D. >> Postdoctoral Fellow >> Okeanos Research Group >> Department of Biological Sciences >> 132 Long Hall >> Clemson University >> Clemson,SC-29634 >> Phone: 864-656-3597 >>Fax: 864-656-0435 >> >> Please note my new email address: > >[hidden email]<mailto:[hidden email]> >> >> >> >> >> >> -- >> >>________________________________________________________________________________ >> Mario M. Moronne, Ph.D. >> >> [hidden email] >> [hidden email] > >*************************** >Tamara Howard >Cell Biology & Physiology >UNM-HSC >Albuquerque, NM >*************************** -- ________________________________________________________________________________ Mario M. Moronne, Ph.D. [hidden email] [hidden email] |
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Neeraj,
Did they do a concentration curve of their primary Ab? Even if working in WB, if used at the same concentration, it will probably be the wrong concentration... I have been using the following protocol with very consistent results in confocal: - PAF 4%, 5 min RT - rinse in PBS 3x 5 min in rotation (cells on coverslips in 6 wells plate) - block in PBS/Triton X100 0.2% (tx)/normal serum 10% (NS) (goat if secondary is from goat) for 15 min in rotation - primary Ab for 1 hr in PBS/Tx/NS at RT (or at 4 degree C overnight), in rotation - rinse in PBS 2x5 min in rotation - block in PBS/Tx/NS 15 min in rotation - secondary Ab 1 hr in PBS/Tx/NS at RT - rinse in PBS - rapid wash in tap water prior to mounting. Hope this will help. Best, Myriam > Date: Thu, 28 Jan 2010 17:35:19 -0500 > From: [hidden email] > Subject: Re: staining nuclear protein > To: [hidden email] > > Thanks Mario, I think the problem they were having was of access as you are pointing out. They tested the antibodies against western blots of nuclear extract where it showed good specificity and a good signal. When they tried doing IF (which is only a couple of times) they got inconsistent staining and weak signal. I am advising them to do series of labeling experiments at different temperatures using variety of fixatives, fixation times and permebalizing agents. > > > Neeraj. > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mario > Sent: Thursday, January 28, 2010 5:09 PM > To: [hidden email] > Subject: Re: staining nuclear protein > > Neeraj, > > Can you define failure? No staining, gross > non-specific staining, lack of expected labeling > morphology? If you don't get any signal then the > antibodies are probably not working. Try new > antibodies. In the nucleus the antibody might be > unable to "find" its target epitope. It might be > comprised through binding to the high > concentrations of nucleic acid, matrix proteins, > etc. Labeling at elevated temperature might help > (37°C) or playing with the pH and elevated ionic > strength or staining for a long time (overnight > in the refrigerator). > > But it does depend on what you mean by failure. > BTW, I have never found BSA to be a very good > blocker, but that is a specificity issue not an > invisibility one. > > Stickiness inside the nucleus translates into a > reduced rate of diffusion. To a first order, the > diffusion constant of a large protein will > decrease in proportion to the apparent > partitioning coefficient of the protein to the > sticky environment it interacts with and depends > on the antibody's charge, etc. So something that > would normally label in 15 minutes could take a > few hours at rm. temperature. > > Mario > > > >Hi Tamara, > > > >I apologize for not including details, my > >colleague is using cultured neurobalstoma cells > >grown as a monolayer on coverslips. > > > > > >Neeraj. > > > > > > > >-----Original Message----- > >From: Confocal Microscopy List > >[mailto:[hidden email]] On > >Behalf Of Tamara A Howard > >Sent: Thursday, January 28, 2010 3:13 PM > >To: [hidden email] > >Subject: Re: staining nuclear protein > > > >I may have missed a post - what kind of samples are these? > >Cultured cells, whole mount tissue/embryos, sections > >(cryo/paraffin/resin)? > > > >Tamara > > > >On Thu, 28 Jan 2010 15:09:42 -0500 > > Neeraj Gohad <[hidden email]> wrote: > >> Hi Mario, > >> > >> This is a new project for my colleague, they have tried > >>using a standard fixation protocol (4% buffered PFA, 0.5% > >>Triton, block with BSA etc..) to no avail. > >> > >> > >> Best, > >> > >> Neeraj. > >> > >> Neeraj V. Gohad, Ph.D. > >> Postdoctoral Fellow > >> Okeanos Research Group > >> Department of Biological Sciences > >> 132 Long Hall > >> Clemson University > >> Clemson,SC-29634 > >> Phone: 864-656-3597 > >>Fax: 864-656-0435 > >> > >> Please note my new email address: > >>[hidden email]<mailto:[hidden email]> > >> > >> > >>From: Confocal Microscopy List > >>[mailto:[hidden email]] On Behalf Of > >>Mario > >> Sent: Thursday, January 28, 2010 1:44 PM > >> To: [hidden email] > >> Subject: staining nuclear protein > >> > >> Neeraj, > >> > >> You don't mention the fixation protocol used. If and > >>when to use Triton (0.2-0.5%; harsh) or Tween (gentler) > >>in part depends on the fixation protocol. Staining > >>nuclear proteins presents its own problems aside from > >>simply permeabilizing the membrane barriers that let your > >>antibodies in. Non-specific labeling is often greatly > >>enhanced because of local ionic strength, pH and very > >>strong electrostatic interactions. You could have a very > >>high affinity antibody (primary and/or secondary) and > >>still end up with diffuse (as in worthless) staining. > >> > >> Cold methanol/acetone fixation might be the way to go if > >>PFA (2-3%) plus Triton (0.5%) doesn't seem to work. Also, > >>staining using higher ionic strength can be helpful and > >>consider conjugating a fluorophore to your primary and > >>forget the secondary altogether. How you block your cells > >>can also be very important. > >> > >> Mario > >> > >> > >> Hi List, > >> > >> One of my colleagues is trying to visualize a nuclear > >>protein using primary antibodies. Is there a special > >>trick or protocol that allows the primary and the > >>fluorescent secondary antibodies better access to > >>proteins inside the nucleus? They have already considered > >>using either Triton or Tween-20. > >> > >> Thanks, > >> > >> Neeraj. > >> > >> Neeraj V. Gohad, Ph.D. > >> Postdoctoral Fellow > >> Okeanos Research Group > >> Department of Biological Sciences > >> 132 Long Hall > >> Clemson University > >> Clemson,SC-29634 > >> Phone: 864-656-3597 > >>Fax: 864-656-0435 > >> > >> Please note my new email address: > > >[hidden email]<mailto:[hidden email]> > >> > >> > >> > >> > >> > >> -- > >> > >>________________________________________________________________________________ > >> Mario M. Moronne, Ph.D. > >> > >> [hidden email] > >> [hidden email] > > > >*************************** > >Tamara Howard > >Cell Biology & Physiology > >UNM-HSC > >Albuquerque, NM > >*************************** > > > -- > ________________________________________________________________________________ > Mario M. Moronne, Ph.D. > > [hidden email] > [hidden email] |
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In reply to this post by Neeraj Gohad-2
Comment posted on behalf of a colleague (Prasanna
Kumar) with considerable experience in staining nuclear proteins: The protocol you use depends on the localization of the protein of interest in nucleus (found in nucleoplasm or a DNA binding etc.) In general I found both Methanol and 4%FA protocols are good enough for most of the proteins with FA protocol maintaining the integrity of DNA/Nucleus better than Methanol. Simultaneous Fixation( with 3.7%FA,4%sucrose) and Permeabilization with 1%Triton X seemed to work well for DNA bound proteins and also different histone modifications. One thing is sure, with the nuclear proteins you need to try all these methods and compare the results as sometimes you see redistribution of the whole pattern and will be wondering which one is the true representative. Good Luck. At 11:35 a.m. 29/01/2010, you wrote: >Thanks Mario, I think the problem they were >having was of access as you are pointing out. >They tested the antibodies against western blots >of nuclear extract where it showed good >specificity and a good signal. When they tried >doing IF (which is only a couple of times) they >got inconsistent staining and weak signal. I am >advising them to do series of labeling >experiments at different temperatures using >variety of fixatives, fixation times and permebalizing agents. > > >Neeraj. > > > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of Mario >Sent: Thursday, January 28, 2010 5:09 PM >To: [hidden email] >Subject: Re: staining nuclear protein > >Neeraj, > >Can you define failure? No staining, gross >non-specific staining, lack of expected labeling >morphology? If you don't get any signal then the >antibodies are probably not working. Try new >antibodies. In the nucleus the antibody might be >unable to "find" its target epitope. It might be >comprised through binding to the high >concentrations of nucleic acid, matrix proteins, >etc. Labeling at elevated temperature might help >(37°C) or playing with the pH and elevated ionic >strength or staining for a long time (overnight >in the refrigerator). > >But it does depend on what you mean by failure. >BTW, I have never found BSA to be a very good >blocker, but that is a specificity issue not an >invisibility one. > >Stickiness inside the nucleus translates into a >reduced rate of diffusion. To a first order, the >diffusion constant of a large protein will >decrease in proportion to the apparent >partitioning coefficient of the protein to the >sticky environment it interacts with and depends >on the antibody's charge, etc. So something that >would normally label in 15 minutes could take a >few hours at rm. temperature. > >Mario > > > >Hi Tamara, > > > >I apologize for not including details, my > >colleague is using cultured neurobalstoma cells > >grown as a monolayer on coverslips. > > > > > >Neeraj. > > > > > > > >-----Original Message----- > >From: Confocal Microscopy List > >[mailto:[hidden email]] On > >Behalf Of Tamara A Howard > >Sent: Thursday, January 28, 2010 3:13 PM > >To: [hidden email] > >Subject: Re: staining nuclear protein > > > >I may have missed a post - what kind of samples are these? > >Cultured cells, whole mount tissue/embryos, sections > >(cryo/paraffin/resin)? > > > >Tamara > > > >On Thu, 28 Jan 2010 15:09:42 -0500 > > Neeraj Gohad <[hidden email]> wrote: > >> Hi Mario, > >> > >> This is a new project for my colleague, they have tried > >>using a standard fixation protocol (4% buffered PFA, 0.5% > >>Triton, block with BSA etc..) to no avail. > >> > >> > >> Best, > >> > >> Neeraj. > >> > >> Neeraj V. Gohad, Ph.D. > >> Postdoctoral Fellow > >> Okeanos Research Group > >> Department of Biological Sciences > >> 132 Long Hall > >> Clemson University > >> Clemson,SC-29634 > >> Phone: 864-656-3597 > >>Fax: 864-656-0435 > >> > >> Please note my new email address: > >>[hidden email]<mailto:[hidden email]> > >> > >> > >>From: Confocal Microscopy List > >>[mailto:[hidden email]] On Behalf Of > >>Mario > >> Sent: Thursday, January 28, 2010 1:44 PM > >> To: [hidden email] > >> Subject: staining nuclear protein > >> > >> Neeraj, > >> > >> You don't mention the fixation protocol used. If and > >>when to use Triton (0.2-0.5%; harsh) or Tween (gentler) > >>in part depends on the fixation protocol. Staining > >>nuclear proteins presents its own problems aside from > >>simply permeabilizing the membrane barriers that let your > >>antibodies in. Non-specific labeling is often greatly > >>enhanced because of local ionic strength, pH and very > >>strong electrostatic interactions. You could have a very > >>high affinity antibody (primary and/or secondary) and > >>still end up with diffuse (as in worthless) staining. > >> > >> Cold methanol/acetone fixation might be the way to go if > >>PFA (2-3%) plus Triton (0.5%) doesn't seem to work. Also, > >>staining using higher ionic strength can be helpful and > >>consider conjugating a fluorophore to your primary and > >>forget the secondary altogether. How you block your cells > >>can also be very important. > >> > >> Mario > >> > >> > >> Hi List, > >> > >> One of my colleagues is trying to visualize a nuclear > >>protein using primary antibodies. Is there a special > >>trick or protocol that allows the primary and the > >>fluorescent secondary antibodies better access to > >>proteins inside the nucleus? They have already considered > >>using either Triton or Tween-20. > >> > >> Thanks, > >> > >> Neeraj. > >> > >> Neeraj V. Gohad, Ph.D. > >> Postdoctoral Fellow > >> Okeanos Research Group > >> Department of Biological Sciences > >> 132 Long Hall > >> Clemson University > >> Clemson,SC-29634 > >> Phone: 864-656-3597 > >>Fax: 864-656-0435 > >> > >> Please note my new email address: > > >[hidden email]<mailto:[hidden email]> > >> > >> > >> > >> > >> > >> -- > >> > >>______________________________________________ > __________________________________ > >> Mario M. Moronne, Ph.D. > >> > >> [hidden email] > >> [hidden email] > > > >*************************** > >Tamara Howard > >Cell Biology & Physiology > >UNM-HSC > >Albuquerque, NM > >*************************** > > >-- >________________________________________________________________________________ >Mario M. Moronne, Ph.D. > >[hidden email] >[hidden email] Dr Barry O'Brien Dept of Biological Sciences, University of Waikato Private Bag 3105 HAMILTON New Zealand Fax 0064 7 838 4324 Phone 0064 7 838 4179 |
 
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F Javier Díez Guerra |
Re: staining nuclear protein
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In reply to this post by Neeraj Gohad-2
Dear Neeraj,
In our experience, the critical point in staining nuclear proteins is to facilitate antibody access to relevant epitopes. You can increase access by extending the incubations times with primary antibody (24 hours and beyond, at room temp.), using Fab fragments (~50kDa) instead of whole antibody molecules (~150kDa for IgGs) or, if you are interested exclusively in the stain of the nucleus, trying controlled permeabilizations of the cells with saponin or digitonin (mild detergents) before fixation. We favoured PFA fixation (2%) over cold methanol or acetone, which tends to collapse cell structures to a higher extent. There is a paper - 19751214[PMID] - with the details of our recent struggles to stain a protein in the nucleus. As a control, we expressed GFP, which localizes within nuclei, and compared the direct (green) fluorescence with the indirect (Vg. red) fluorescence obtained by immunocytochemistry. Hope it helps -- F Javier Díez Guerra, PhD Profesor Titular Centro de Biología Molecular Severo Ochoa C/ Nicolás Cabrera, 1 Universidad Autónoma Ctra Colmenar Viejo Km 15 Cantoblanco, 28049 Madrid SPAIN phone: +34 91 196 4612 e-mail: [hidden email] |
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In reply to this post by Mario-2
Many
Thanks to everyone who replied to my staining of nuclear proteins post. I have
compiled the suggestions and protocols of the folks who replied. Best
Regards and have a Good Weekend! Neeraj.
Neeraj V. Gohad, Ph.D. Postdoctoral Fellow Okeanos Research Group Department of Biological Sciences 132 Long Hall Clemson University Clemson,SC-29634 Phone: 864-656-3597 Fax: 864-656-0435 Please note my new email address: [hidden email] 1)
The
fixation, not membrane permeabilization, seems to be more of a factor for
nuclear proteins.
Try Methanol (100% formaldehyde-free at -20C for 20 min). Compare with 3.7% FA
at RT.
Contact me off-list if you need further details.
Besrt Wishes,
Fred E. Indig, Ph.D.
Head, Confocal Imaging Facility 2)
You
don't mention the fixation protocol used. If and when to use Triton (0.2-0.5%;
harsh) or Tween (gentler) in part depends on the fixation protocol. Staining
nuclear proteins presents its own problems aside from simply permeabilizing the
membrane barriers that let your antibodies in. Non-specific labeling is often
greatly enhanced because of local ionic strength, pH and very strong
electrostatic interactions. You could have a very high affinity antibody
(primary and/or secondary) and still end up with diffuse (as in worthless)
staining. Cold methanol/acetone fixation might be the
way to go if PFA (2-3%) plus Triton (0.5%) doesn't seem to work. Also, staining
using higher ionic strength can be helpful and consider conjugating a
fluorophore to your primary and forget the secondary altogether. How you block
your cells can also be very important. Mario 3)
I
used to have pretty good success accessing DNA binding proteins using 0.5%
triton for about 15minutes.Cold MetOH fixation also worked well... Good
luck! Danielle Crippen Morphology and Imaging Core Manager Buck
Institute for Age Research 4)
Can
you define failure? No staining, gross non-specific staining, lack of expected
labeling morphology? If you don't get any signal then the antibodies are
probably not working. Try new antibodies. In the nucleus the antibody might be
unable to "find" its target epitope. It might be comprised through
binding to the high concentrations of nucleic acid, matrix proteins, etc.
Labeling at elevated temperature might help (37°C) or playing with the pH and elevated
ionic strength or staining for a long time (overnight in the refrigerator). But
it does depend on what you mean by failure. BTW, I have never found BSA to be a
very good blocker, but that is a specificity issue not an invisibility one. Stickiness
inside the nucleus translates into a reduced rate of diffusion. To a first
order, the diffusion constant of a large protein will decrease in proportion to
the apparent partitioning coefficient of the protein to the sticky environment
it interacts with and depends on the antibody's charge, etc. So something that
would normally label in 15 minutes could take a few hours at rm. temperature. Mario 5)
Did
they do a concentration curve of their primary Ab? Even if working in WB, if
used at the same concentration, it will probably be the wrong concentration...
I have been using the following protocol with very consistent results in
confocal: 6)
Comment
posted on behalf of a colleague (Prasanna Kumar) with considerable experience
in staining nuclear proteins: The protocol you use depends on the
localization of the protein of interest in nucleus (found in nucleoplasm or a
DNA binding etc.) In general I found both Methanol and 4%FA protocols are good
enough for most of the proteins with FA protocol maintaining the integrity of
DNA/Nucleus better than Methanol. Simultaneous Fixation( with3.7%FA,4%sucrose)
and Permeabilization with 1%Triton X seemed to work well for DNA bound proteins
and also different histone modifications. One thing is sure, with the nuclear
proteins you need to try all these methods and compare the results as sometimes
you see redistribution of the whole pattern and will be wondering which one is
the true representative. Good Luck. Barry O'Brien 7)
In
our experience, the critical point in staining nuclear proteins is to
facilitate antibody access to relevant epitopes. You can increase access by
extending the incubations times with primary antibody (24 hours and beyond, at
room temp.), using Fab fragments (~50kDa) instead of whole antibody molecules
(~150kDa for IgGs) or, if you are interested exclusively in the stain of the
nucleus, trying controlled permeabilizations of the cells with saponin or
digitonin (mild detergents) before fixation. We favoured PFA fixation (2%) over cold
methanol or acetone, which tends to collapse cell structures to a higher
extent. There is a paper - 19751214[PMID] - with the details of our recent
struggles to stain a protein in the nucleus. As a control, we expressed GFP,
which localizes within nuclei, and compared the direct (green) fluorescence
with the indirect (Vg. red) fluorescence obtained by immunocytochemistry. Hope it helps F Javier Díez Guerra, PhD Profesor Titular |
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