Overview

  • Product name
  • Description
    Rabbit polyclonal to NUMB
  • Host species
    Rabbit
  • Specificity
    The antibody may cross react with NUMBL protein (Swissprot ID Q9Y6R0) because the immunogen used to raise this antibody has a 92% homology with NUMBL protein from Human and Mouse. Denaturing of the protein sample is imperative to the success of ab14140 detecting a single specific band in WB (denature at 95°C for 15min). An additional, higher band (around 80kDa) is seen if not denatured.
  • Tested applications
    Suitable for: IHC-P, ICC/IF, IHC-Fr, WB, IP, IHC - Wholemountmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Zebrafish
    Predicted to work with: Chicken, Monkey, Non human primates
  • Immunogen

    Synthetic peptide derived from residues 600 to the C-terminus of Human NUMB.

    Read Abcam's proprietary immunogen policy .

  • Positive control
    • WB: Mouse and Rat Brain Tissue Lysates. IHC-P: Human adrenal gland tissue IHC-Fr: Rat Brain tissue ICC/IF: Mouse neocortex neural precursor cells

Properties

Applications

Our Abpromise guarantee covers the use of ab14140 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use at an assay dependent concentration.
ICC/IF Use a concentration of 8 µg/ml.
IHC-Fr Use at an assay dependent concentration.
WB Use a concentration of 0.5 - 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 70 kDa).Can be blocked with Human NUMB peptide (ab14141).
IP Use at an assay dependent concentration.
IHC - Wholemount 1/500.

Target

  • Function
    Plays a role in the process of neurogenesis. Required throughout embryonic neurogenesis to maintain neural progenitor cells, also called radial glial cells (RGCs), by allowing their daughter cells to choose progenitor over neuronal cell fate. Not required for the proliferation of neural progenitor cells before the onset of neurogenesis. Also involved postnatally in the subventricular zone (SVZ) neurogenesis by regulating SVZ neuroblasts survival and ependymal wall integrity. May also mediate local repair of brain ventricular wall damage.
  • Sequence similarities
    Contains 1 PID domain.
  • Post-translational
    modifications
    Isoform 1 and isoform 2 are ubiquitinated by LNX leading to their subsequent proteasomal degradation (By similarity). Ubiquitinated; mediated by SIAH1 and leading to its subsequent proteasomal degradation.
  • Cellular localization
    Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • c14_5527 antibody
    • C14orf41 antibody
    • FLJ31314 antibody
    • h Numb antibody
    • h-Numb antibody
    • Numb antibody
    • Numb homolog (Drosophila) antibody
    • Numb homolog antibody
    • Numb protein homolog antibody
    • NUMB_HUMAN antibody
    • Protein numb homolog antibody
    • Protein S171 antibody
    • S171 antibody
    see all

Images

  • Image courtesy of Human Protein Atlas

    ab14140 staining NUMB in Human adrenal gland. The paraffin embedded human tissue was incubated with ab14140 (1/250 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. Ab14140 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.

    Further results for this antibody can be found at www.proteinatlas.org

  • NUMB was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 5µg of Rabbit polyclonal to NUMB and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain whole tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab14140.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 75kDa: NUMB.
  • ab14140 detecting NUMB protein in dissociated neural precursor cells cultured from embryonic day 13 mouse neocortex in the presence of bFGF, EGF and LIF. NUMB immunoreactivity (green) was found in the cytosol of neural precursor cells. This localisation is in general agreement with published studies (e.g. Chen et al., EJN, 2005), where similar labelling is observed in the cytosol of cells of the grey matter of adult mouse spinal cord. Immunocytochemistry: All steps were performed in PBS. Cells were fixed in 4% PFA for 15min, permeabilised with 0.1% TX100 for 10min and blocked with 5% BSA, 0.1% TX100 for 45min. ab14140 was incubated at 8µg/ml for 12h at 4°C in 5% BSA, 0.1% TX100. Cultures were washed (3x) of primary antibody solution. Goat anti-rabbit AlexaFluor 488 was used as secondary antibody (1/400) in 5% BSA, 0.1% TX100 for 1h at RT. To-pro-3 was used as a nuclear counterstain (blue). Treated cultures were mounted on glass coverslips with Mowiol.

  • ab14140 at 1/100 staining rat brain tissue sections by IHC-Fr. The tissue was paraformaldehyde fixed and blocked with 5% serum prior to incubation with the antibody for 24 hours. An Alexa-Fluor ® 488 conjugated goat anti-rabbit antibody was used as the secondary. NUMB staining is shown in green.

    See Abreview

  • All lanes : Anti-NUMB antibody (ab14140) at 1 µg/ml

    Lane 1 : Brain (Rat) Tissue Lysate
    Lane 2 : Brain (Mouse) Tissue Lysate
    Lane 3 : Human brain tissue lysate - total protein (ab29466)

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Anti-Rabbit IgG VHH Single Domain (HRP) (ab191866) at 1/50000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 70 kDa
    Observed band size: 75 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 74 kDa (possible isoform), 77 kDa (possible post-translational modification)


    Exposure time: 4 minutes


    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab14140 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

  • Anti-NUMB antibody (ab14140) at 0.5 µg/ml + Adult Rat whole brain lysate at 40 µg

    Secondary
    Goat anti-rabbit IgG HRP at 1/20000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 70 kDa
    Observed band size: 75 kDa why is the actual band size different from the predicted?


    Exposure time: 30 seconds


     Denaturing of the protein sample was imperative to the success of this WB (denatured at 95°C for 15min). An additional, higher band (around 80kDa) is seen if not denatured. Original concentration tried was 1ug/ml but we found this high given the circumstances and have since used 0.5ug/ml. Theoretically, even less could be used.

  • ab14140 staining NUMB in Zebrafish embryo nervous system by Immunohistochemistry (Whole mount). Samples were incubated with primary antibody (1/500 in BSA blocking buffer) for 48 hours at 4°C. An Alexa Fluor®488-conjugated Goat anti-rabbit  polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • Anti-NUMB antibody (ab14140) at 0.5 µg/ml + Mouse whole brain lysate

    Secondary
    Goat anti-rabbit IgG HRP at 1/20000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 70 kDa
    Observed band size: 75 kDa why is the actual band size different from the predicted?


    Exposure time: 30 seconds


    Denaturing of the protein sample was imperative to the success of this WB. An additional, higher band (around 80kDa) is seen if not denatured. Original concentration tried was 1ug/ml but we found this high given the circumstances and have since used 0.5ug/ml. Theoretically, even less could be used.
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References

This product has been referenced in:
  • Zhang Z  et al. Nrf2 antioxidant pathway suppresses Numb-mediated epithelial-mesenchymal transition during pulmonary fibrosis. Cell Death Dis 9:83 (2018). WB . Read more (PubMed: 29362432) »
  • Iannolo G  et al. MiR34 inhibition induces human heart progenitor proliferation. Cell Death Dis 9:368 (2018). Read more (PubMed: 29511160) »
See all 32 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
IHC - Wholemount
Sample
Zebrafish Embryo (Nervous system)
Specification
Nervous system

Abcam user community

Verified customer

Submitted Nov 03 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Pancreas)
Specification
Pancreas
Fixative
Methanol
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 20°C

Abcam user community

Verified customer

Submitted May 07 2013

Answer

Thank you for your email.

We had sent you the following information based on your inquiry about the homology between NUMB and NUMBL:

The overall homology between human NUMB and NUMBL proteins is only 42-67%.

But for the immunogen sequence, the homology is 93%, unfortunately. Thus, crossreactivity is likely and we have updated the datasheet with this information.

The immunogen is a synthetic peptide derived from residues 600 to the C-terminus of Human NUMB. The exact immunogen sequence is proprietary and we are not able to provide it in this case.

I hope this information is nevertheless helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Thank you for contacting us.

The overall homology between human NUMB and NUMBL proteins is only 42-67%.

But for the immunogen sequence, the homology is 93%, unfortunately. Thus, crossreactivity is likely. I'll have thelab look into this further.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Question
Answer

Thank you for contacting us.

Could you please let me know the NUMBL sequence or the according database entry so that I can check sequence homology for between NUMB and NUMBL in the immunogen region?

Thank you for your help!

I look forward to hear back from you.

Read More
Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Chicken Tissue sections (Spinal cord)
Specification
Spinal cord
Fixative
Paraformaldehyde
Antigen retrieval step
None
Permeabilization
Yes - 0.1% Triton-X-100
Blocking step
Serum as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 20°C

David Rousso

Verified customer

Submitted Sep 09 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (embryo brain)
Specification
embryo brain
Fixative
Paraformaldehyde
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 6%

Abcam user community

Verified customer

Submitted May 22 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (Brain)
Specification
Brain
Fixative
Paraformaldehyde
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Mr. Darius Gleason

Verified customer

Submitted Feb 01 2007

Answer

Thank you for your patience. Again, I'm sorry to hear you are having a problem with ab14955. I will do my best to troubleshoot with you. In my last e-mail, I suggested the following modifications to your protocol: 1) Reduce primary antibody - try 1:1000 dilution in blocking solution 2) Reduce incubation time in primary antibody - try 1-3hrs at 4C 3) Use goat serum at 10% if not already doing so. In addition to the above, I would also suggest: 4) Reduce secondary antibody - try 1:500 5) Contact Kirk McManus for his protocol and about his ICC image. Could you also provide the following information regarding your protocol: 1) % paraformaldehyde 2) % Triton X-100 3) images of staining To achieve exceptional levels of quality for our customers, we post everything we know about each product. In the case of ab14955, this antibody has not been tested in IHC. Although some companies don't use the IHC/ICC distinction, there is definitely a difference between staining tissues and cells. While some antibodies might work in both applications, others work only in one. For this reason, we specify whether an antibody has been successfully tested in ICC and/or ICH. I see your point on the use of "IF" but this only specifies the detection method (how the 2nd antibody is conjugated). It is true that antibodies have worked in applications that have not been tested by Abcam and when we are aware of this, we update the datasheet to inform the customers. In regards to the image from Kirk, I've asked one of my colleagues to examine the image. Histone H3 is phosphorylated during mitosis. This is reflected in the image submitted by Kirk McManus, I think that the datasheet is not bad considering this and is consistent with this; showing staining of the condensed chromosomes but not the interphase cells. I suggest that you contact him for additional information. Could you also tell me how one would capture the cells in mitosis in tissue preparations. This may be why you have not been able to find staining as histone H3 serine 10 phosphorylation is very low when performing immunostaining outside of mitosis. I hope this information will help improve your IHC experiments. Please don't hesitate to contact us for further advice.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - whole (Whole brain lysate - adult)
Loading amount
40 µg
Specification
Whole brain lysate - adult
Treatment
Denatured at 95C for 15min
Blocking step
Milk as blocking agent for 20 minute(s) · Concentration: 5%

Dr. Randal Moldrich

Verified customer

Submitted May 18 2006

1-10 of 12 Abreviews or Q&A

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