Re: [Histonet] Human nuclear antibody MAB1281
I had/have the same problem with that ab on FFPWS +/- AR Also negative, under same conditions: Serotec's 6970-1257, Abcam's ab5675 I use 2 abs on hu teratomas in mouse host: Takara clontech STEM121 antibody Abcam anti MTCO2 ab110258 Images to be seen on Histonet Images site Carl Hobbs FIBMS Histology and Imaging Manager Wolfson CARD Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6813 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Human nuclear antibody MAB1281
Liz, Any chance you could find out what protocol was used by the ones that have used it? or help me find some of the literature references? Everywhere I look I just see people who have not gotten it to work asking for help. Thanks, Eva On Thu, Feb 25, 2016 at 10:39 AM, Elizabeth Chlipala <l...@premierlab.com> wrote: > Eva > > We have worked with various antibodies in order to detect human cells in a > rodent background. I know that this antibody has been referenced in the > literature and I also have known of other individuals who have had success > with it, but in our hands we have never been able to make it work on FFPE > tissues but I believe it works in frozen sections. We have opted to try > some different antibodies. > > NuMA -we researched this protein and determined that it may work -here is > a snippet of what we found from a quick look into the Human Protein Atlas > entry for NUMA1. Though it is functionally involved in the structural > rearrangement of DNA during mitosis and apoptosis, it is a constitutively > expressed structural protein. From the images on HPA ( > http://www.proteinatlas.org/ENSG0137497-NUMA1/tissue/primary+data) > it looks like it is almost universally expressed, with the one exception of > hepatocytes. The vast majority of neurons, which are overwhelmingly > post-mitotic, show fairly strong expression, so that's encouraging. We > initially looked at the cell signaling antibody (our target was human cells > in a rat background) but found that it does cross react with rat, its fine > with mouse but this antibody is our hands was found to be sensitive to time > in fixation and retrieval time and temp. We opted to try another source > for this antibody - abcam. They had several different antibodies for that > target. We work with their antibodies a lot and they normally do not list > the sequence of the antigen so we could not check the sequence homology to > other species ourselves but you can call them and they will provide that > information. Here is what they provided to us - "We have not tested > ab86129 for cross reactivity with rat and we have not received any > researcher feedback for using the antibody with this species. The immunogen > sequence has 56% identity with the rat protein, so it is unlikely that it > will cross react. However we have not confirmed this experimentally and we > do not know if it would show non-specific staining with rat. I do think > ab86129 would be the best antibody to try since it has the lowest homology > with the rat protein. The other antibodies that have not been tested with > rat have 69% (ab55767) or 88% (ab84680) homology." Ab86129 worked very > nicely in our hands. > > HLA-1 this will also work nicely but the protein is expressed in the > cytoplasm and expression levels can vary but it does work nicely in some > instances. > > So for us its dependent upon the project as to which antibody we will use > but the NuMA is expressed in the nuclei so it will work quite nicely for > dual staining of another protein that is expressed in the cytoplasm or cell > membrane. > > Good Luck > > Liz > > Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC > Premier Laboratory, LLC > PO Box 18592 > Boulder, CO 80308 > (303) 682-3949 office > (303) 682-9060 fax > (303) 881-0763 cell > l...@premierlab.com > www.premierlab.com > > Ship to Address: > > Premier Laboratory, LLC > 1567 Skyway Drive, Unit E > Longmont, CO 80504 > > -Original Message- > From: Eva Permaul via Histonet [mailto:histonet@lists.utsouthwestern.edu] > Sent: Thursday, February 25, 2016 7:07 AM > To: histonet > Subject: [Histonet] Human nuclear antibody MAB1281 > > Good morning, > We recently bought MAB1281 with the hope of being able to determine if the > cells in a mouse model was human or mouse. > We want to use it to stain FFPE tissues. I have tried it with Citrate per > the companies recommendation. I ran it on a human tonsil but saw no > staining at all. > Is there anyone who has been able to get this antibody to work? And if so > would you please share your protocol? > Thank you, > Eva > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Human nuclear antibody MAB1281
Eva We have worked with various antibodies in order to detect human cells in a rodent background. I know that this antibody has been referenced in the literature and I also have known of other individuals who have had success with it, but in our hands we have never been able to make it work on FFPE tissues but I believe it works in frozen sections. We have opted to try some different antibodies. NuMA -we researched this protein and determined that it may work -here is a snippet of what we found from a quick look into the Human Protein Atlas entry for NUMA1. Though it is functionally involved in the structural rearrangement of DNA during mitosis and apoptosis, it is a constitutively expressed structural protein. From the images on HPA (http://www.proteinatlas.org/ENSG0137497-NUMA1/tissue/primary+data) it looks like it is almost universally expressed, with the one exception of hepatocytes. The vast majority of neurons, which are overwhelmingly post-mitotic, show fairly strong expression, so that's encouraging. We initially looked at the cell signaling antibody (our target was human cells in a rat background) but found that it does cross react with rat, its fine with mouse but this antibody is our hands was found to be sensitive to time in fixation and retrieval time and temp. We opted to try another source for this antibody - abcam. They had several different antibod ies for that target. We work with their antibodies a lot and they normally do not list the sequence of the antigen so we could not check the sequence homology to other species ourselves but you can call them and they will provide that information. Here is what they provided to us - "We have not tested ab86129 for cross reactivity with rat and we have not received any researcher feedback for using the antibody with this species. The immunogen sequence has 56% identity with the rat protein, so it is unlikely that it will cross react. However we have not confirmed this experimentally and we do not know if it would show non-specific staining with rat. I do think ab86129 would be the best antibody to try since it has the lowest homology with the rat protein. The other antibodies that have not been tested with rat have 69% (ab55767) or 88% (ab84680) homology." Ab86129 worked very nicely in our hands. HLA-1 this will also work nicely but the protein is expressed in the cytoplasm and expression levels can vary but it does work nicely in some instances. So for us its dependent upon the project as to which antibody we will use but the NuMA is expressed in the nuclei so it will work quite nicely for dual staining of another protein that is expressed in the cytoplasm or cell membrane. Good Luck Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell l...@premierlab.com www.premierlab.com Ship to Address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 -Original Message- From: Eva Permaul via Histonet [mailto:histonet@lists.utsouthwestern.edu] Sent: Thursday, February 25, 2016 7:07 AM To: histonet Subject: [Histonet] Human nuclear antibody MAB1281 Good morning, We recently bought MAB1281 with the hope of being able to determine if the cells in a mouse model was human or mouse. We want to use it to stain FFPE tissues. I have tried it with Citrate per the companies recommendation. I ran it on a human tonsil but saw no staining at all. Is there anyone who has been able to get this antibody to work? And if so would you please share your protocol? Thank you, Eva ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Human nuclear antibody MAB1281
Good morning, We recently bought MAB1281 with the hope of being able to determine if the cells in a mouse model was human or mouse. We want to use it to stain FFPE tissues. I have tried it with Citrate per the companies recommendation. I ran it on a human tonsil but saw no staining at all. Is there anyone who has been able to get this antibody to work? And if so would you please share your protocol? Thank you, Eva ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
