Overview

  • Product name
  • Description
    Rabbit polyclonal to Nucleolin
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IP, IHC-Fr, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Nucleolin.

    Read Abcam's proprietary immunogen policy (Peptide available as ab25315.)

  • Positive control
    • HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate, Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate, A431 (Human epithelial carcinoma cell line) Whole Cell Lysate, MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate, Brain (Mouse) Tissue Lysate, Pancreas (Mouse) Tissue Lysate, PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab22758 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
WB Use a concentration of 1 µg/ml. Detects a band of approximately 76 kDa (predicted molecular weight: 76 kDa).
IP Use at an assay dependent concentration.
IHC-Fr 1/400.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ICC/IF Use a concentration of 1 µg/ml.

Target

  • Function
    Nucleolin is the major nucleolar protein of growing eukaryotic cells. It is found associated with intranucleolar chromatin and pre-ribosomal particles. It induces chromatin decondensation by binding to histone H1. It is thought to play a role in pre-rRNA transcription and ribosome assembly. May play a role in the process of transcriptional elongation. Binds RNA oligonucleotides with 5'-UUAGGG-3' repeats more tightly than the telomeric single-stranded DNA 5'-TTAGGG-3' repeats.
  • Sequence similarities
    Contains 4 RRM (RNA recognition motif) domains.
  • Post-translational
    modifications
    Some glutamate residues are glycylated by TTLL8. This modification occurs exclusively on glutamate residues and results in a glycine chain on the gamma-carboxyl group.
  • Cellular localization
    Nucleus > nucleolus. Cytoplasm. Localized in cytoplasmic mRNP granules containing untranslated mRNAs.
  • Information by UniProt
  • Database links
  • Alternative names
    • C23 antibody
    • FLJ45706 antibody
    • MS1116 antibody
    • NCL antibody
    • Nucl antibody
    • NUCL_HUMAN antibody
    • Nucleolin antibody
    • Protein C23 antibody
    see all

Images

  • ICC/IF image of ab22758 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab22758, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

  • IHC image of Nucleolin staining in human tonsil FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab22758, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • All lanes : Anti-Nucleolin antibody (ab22758) at 1 µg/ml

    Lane 1 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 2 : Brain (Mouse) Tissue Lysate
    Lane 3 : Pancreas (Mouse) Tissue Lysate
    Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Predicted band size: 76 kDa
    Observed band size: 100 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 60 kDa. We are unsure as to the identity of these extra bands.

  • Nucleolin was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to Nucleolin 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, Jurkat whole cell extract 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 ab22758.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 100kDa: Nucleolin.
  • Lane 1 : Marker
    Lanes 2-5 : Anti-Nucleolin antibody (ab22758) at 1 µg/ml

    Lane 2 : Jurkat whole cell lysate (ab7899) at 20 µg
    Lane 3 : A431 whole cell lysate (ab7909) at 20 µg
    Lane 4 : Jurkat whole cell lysate (ab7899) at 20 µg with Human Nucleolin peptide (ab25315) at 1 µg/ml
    Lane 5 : A431 whole cell lysate (ab7909) at 20 µg with Human Nucleolin peptide (ab25315) at 1 µg/ml

    Secondary
    Lanes 2-5 : Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 dilution


    Performed under reducing conditions.

    Predicted band size: 76 kDa
    Observed band size: 100 kDa why is the actual band size different from the predicted?

  • ab22758 staining nucleolin in HeLa cells treated with Triptolide from Tripterygium wilfordii (ab120720), by ICC/IF. Changes in nuclear localization of nucleolin (from nuclelous to whole nucleous) correlates with increased concentration of Triptolide from Tripterygium wilfordii, as described in literature.
    The cells were incubated at 37°C for 1h in media containing different concentrations of ab120720 (Triptolide from Tripterygium wilfordii ) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab22758 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody.

References

This product has been referenced in:
  • Zhang S  et al. Dual radiosensitization and anti-STAT3 anti-proliferative strategy based on delivery of gold nanoparticle - oligonucleotide nanoconstructs to head and neck cancer cells. Nanotheranostics 2:1-11 (2018). Read more (PubMed: 29291159) »
  • Telechea-Fernández M  et al. New localization and function of calpain-2 in nucleoli of colorectal cancer cells in ribosomal biogenesis: effect of KRAS status. Oncotarget 9:9100-9113 (2018). Read more (PubMed: 29507677) »
See all 62 Publications for this product

Customer reviews and Q&As

1-10 of 15 Abreviews or Q&A

Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (U2OS cells)
Total protein in input
500 µg
Immuno-precipitation step
Protein A/G
Specification
U2OS cells

Abcam user community

Verified customer

Submitted May 12 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (MEF cells)
Gel Running Conditions
Reduced Denaturing (4-12% Gradient Gel)
Loading amount
20 µg
Specification
MEF cells
Blocking step
Licor Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 28 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (U2OS cells)
Gel Running Conditions
Reduced Denaturing (4-12% Gradient Gel)
Loading amount
50 µg
Specification
U2OS cells
Blocking step
Licor Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 05 2016

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (SHSY-5Y)
Permeabilization
No
Specification
SHSY-5Y
Fixative
Methanol

Abcam user community

Verified customer

Submitted Aug 21 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Background buster as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C
Sample
Human Cell (Ovarian cancer cell line)
Specification
Ovarian cancer cell line
Permeabilization
Yes - 0.25% tritonx X-100
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jun 02 2014

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3%
Sample
Fruit fly (Drosophila melanogaster) Cell (adult testis)
Specification
adult testis
Permeabilization
Yes - .1% TritonX1000
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jul 10 2013

Question
Answer

Vielen Dank für Ihren Anruf.

Leider handelt handelt es sich bei dem monoklonen Antikörper hierbei nicht um ein In-Haus Produkt, so dass wir das Hybridoma nicht weiterverkaufen können. Des weiteren darf ich Ihnen aus betrieblichen Gründen nicht den Hersteller dieses Antikörpers nennen.

Die gute Nachricht ist, dass sich der "Montags-Schleier" verzogen hat, und sich die Bulk-Order Seite fast wie von alleine auffinden ließ:

https://www.abcam.com/index.html?pageconfig=resource&rid=14647

Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Benutzen Sie unsere Produkte? Schicken Sie uns einen Abreview. Verdienen Sie sich eine Belohnung!
https://www.abcam.com/abreviews

Read More

Answer

Thank you again for letting us know about this problem and for your patience while our lab is working with this antibody.
My colleagues in the lab have re-tested the antibody in mouse lung tissue, and we have also found that the apical staining is missing when using the most recent lot that you've used. We are currently running a couple of more tests to make sure this can not be optimized. It is now part of our QC criteria for new lots of this antibody to show the apical staining, so this should reduce the lot variability in the future. The lab is also currently testing other lots of the antibody to see if any produce the apical staining, and I will update you as soon as we know whether we have a good lot to send you.
Please let me know if there is anything else that we can do for you, and thank you again for your patience.

Read More

Answer

Thank you for contacting us and your interest in our products.

As you have seen from the datasheet of the Anti-Nucleolin antibody (ab22758) we observe a consistent band of ˜100 kDa when performing Western blotting or immunoprecipitaion. We have not performed siRNA knockdown in order to confirm that the band detected is as a result of nucleolin expression but the results we have observed are consistent with research carried out on the protein as can be reviewed from the following journal articles:

http://www.nature.com/nm/journal/v17/n9/abs/nm.2444.html?lang=en?WT.ec_id=NM-201109

http://www.biochemj.org/bj/289/0109/2890109.pdf

http://jcs.biologists.org/content/112/6/761.full.pdf

Although the amino acid structure of the protein would predict a size of 76 kDa, it goes through a significant amount of post-translational modification including methylation andphosphorylation which increases the actual size of the protein.

The band we have observed with the antibody, as well as the strong specific staining withinthe human HeLa cells shows that the antibody is capable of binding the Nucleolin protein.

I hope this information has been of help. If you have any further questions, please do not hesitate to ask.

Read More

Answer

Thank you very much for your interest in ab22758.

Because we carry over 70,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.


To our knowledge, ab22758 has not been tested in zebrafish.

Therefore, I can offer a discount off a future purchase if you buy ab22758 now, test it in zebrafish and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of ab22758 off a future order.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab22758 in zebrafish. I will then send a discount code. This code must be issued before purchasing ab22758 so please wait for my reply before ordering.

2. Purchase ab22758 either by phone, fax, or online (www.abcam.com).

3. Test it in zebrafish.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: https://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any product ordered and the discount code is valid for 4 months after issue.
Please remember that submission of the Abreview is sufficient for the discount code to become active.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab22758 turns out to be unsuitable for zebrafish, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

Read More

1-10 of 15 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up