Overview

  • Product name
    Anti-ErbB2 Affibody® Molecule (FITC)
    See all ErbB 2 affibody® molecule
  • Conjugation
    FITC. Ex: 493nm, Em: 528nm
  • Conjugation notes
    This molecule is conjugated at the unique C-terminal cysteine using a maleimide activated fluorescin reagent. The conjugated Anti-ErbB2 Affibody® molecule is excellent as a one step reagent for fluorescence studies of ErbB2 expression on cells and frozen tissue sections and for flow cytometry.
  • Specificity
    This product binds to the extracellular domain of human ErbB2.
  • Tested applications
    Suitable for: Flow Cyt, IHC-Fr, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    -

  • General notes

    This product is a recombinant protein produced in E.coli.

    What are Affibody Molecules?
    Affibody® affinity ligands are unique research reagents, produced using innovative protein-engineering technologies. They are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold has excellent features as an affinity ligand and can be designed to bind with high affinity to any given target protein. The domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants. Thus, the libraries consist of a multitude of protein ligands with an identical backbone and variable surface-binding properties. In function, Affibody® Molecules mimic monoclonal antibodies. Compared to antibodies, the most striking dissimilarity of Affibody® Molecules is the small size. Affibody® Molecules have a molecular weight of 6kDa, compared to the molecular weight of antibodies, which is 150kDa. In spite of its small size, the binding site of Affibody® Molecules is similar to that of an antibody. The advantages of Affibody® Molcules over antibodies are: -their small size -the simple structure of the molecules -its robust physical properties; able to withstand a broad range of analytical conditions, including extreme pH and elevated temperature -its ability to fold correctly intracellularly -the fast and cost effective production in bacteria -the potential to couple Affibody® Molecules in multimeric constructs Affibody® Molecules have highly competitive properties for applications within affinity purification, sample preparation, protein detection and in vitro diagnostics.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term.
  • Storage buffer
    pH: 7.20
    Preservative: 0.02% Sodium azide
    Constituents: 0.328% Sodium phosphate, 0.87% Sodium chloride
  • Concentration information loading...
  • Purification notes
    The purity of this product is >98% as determined by RP-HPLC analysis.
  • Research areas
  • Function
    Protein tyrosine kinase that is part of several cell surface receptor complexes, but that apparently needs a coreceptor for ligand binding. Essential component of a neuregulin-receptor complex, although neuregulins do not interact with it alone. GP30 is a potential ligand for this receptor. Regulates outgrowth and stabilization of peripheral microtubules (MTs). Upon ERBB2 activation, the MEMO1-RHOA-DIAPH1 signaling pathway elicits the phosphorylation and thus the inhibition of GSK3B at cell membrane. This prevents the phosphorylation of APC and CLASP2, allowing its association with the cell membrane. In turn, membrane-bound APC allows the localization of MACF1 to the cell membrane, which is required for microtubule capture and stabilization.
    In the nucleus is involved in transcriptional regulation. Associates with the 5'-TCAAATTC-3' sequence in the PTGS2/COX-2 promoter and activates its transcription. Implicated in transcriptional activation of CDKN1A; the function involves STAT3 and SRC. Involved in the transcription of rRNA genes by RNA Pol I and enhances protein synthesis and cell growth.
  • Tissue specificity
    Expressed in a variety of tumor tissues including primary breast tumors and tumors from small bowel, esophagus, kidney and mouth.
  • Involvement in disease
    Hereditary diffuse gastric cancer
    Glioma
    Ovarian cancer
    Lung cancer
    Gastric cancer
    Chromosomal aberrations involving ERBB2 may be a cause gastric cancer. Deletions within 17q12 region producing fusion transcripts with CDK12, leading to CDK12-ERBB2 fusion leading to truncated CDK12 protein not in-frame with ERBB2.
  • Sequence similarities
    Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Autophosphorylated. Autophosphorylation occurs in trans, i.e. one subunit of the dimeric receptor phosphorylates tyrosine residues on the other subunit (Probable). Ligand-binding increases phosphorylation on tyrosine residues (PubMed:27134172). Signaling via SEMA4C promotes phosphorylation at Tyr-1248 (PubMed:17554007). Dephosphorylated by PTPN12 (PubMed:27134172).
  • Cellular localization
    Cytoplasm. Nucleus and Cell membrane. Cytoplasm, perinuclear region. Nucleus. Translocation to the nucleus requires endocytosis, probably endosomal sorting and is mediated by importin beta-1/KPNB1.
  • Information by UniProt
  • Alternative names
    • Verb b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog
    • C erb B2/neu protein
    • CD340
    • CD340 antigen
    • Cerb B2/neu protein
    • CerbB2
    • Erb b2 receptor tyrosine kinase 2
    • ERBB2
    • ERBB2_HUMAN
    • HER 2
    • HER 2/NEU
    • HER2
    • Herstatin
    • Human epidermal growth factor receptor 2
    • Metastatic lymph node gene 19 protein
    • MLN 19
    • MLN19
    • NEU
    • NEU proto oncogene
    • Neuro/glioblastoma derived oncogene homolog
    • Neuroblastoma/glioblastoma derived oncogene homolog
    • NGL
    • p185erbB2
    • Proto-oncogene c-ErbB-2
    • Proto-oncogene Neu
    • Receptor tyrosine-protein kinase erbB-2
    • TKR1
    • Tyrosine kinase type cell surface receptor HER2
    • Tyrosine kinase-type cell surface receptor HER2
    • V erb b2 avian erythroblastic leukemia viral oncogene homolog 2
    • V erb b2 avian erythroblastic leukemia viral oncogene homolog 2 (neuro/glioblastoma derived oncogene homolog)
    • V erb b2 avian erythroblastic leukemia viral oncoprotein 2
    • V erb b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog
    • V erb b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)
    • Verb b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab31891 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
Flow Cyt Use at an assay dependent dilution.
IHC-Fr Use at an assay dependent dilution.
Staining of paraffin embedded tissues is not recommended. AFFIBODY® MOLECULES ARE PROTOCOL SPECIFIC. PLEASE REFER TO THE "PROTOCOLS" LINKS BELOW.
ICC/IF Use at an assay dependent dilution.

Images

  • Human mammary gland cell line SK-BR3 cells were stained with fluorescin conjugated Anti-ErbB2 Affibody® molecule (ab31891). The staining was localized to the membrane of the ErbB2 expressing human mammary gland cells. Nuclei were counter stained with DAPI (blue fluorescence). The SK-BR3 cells were stained for 30 minutes at a concentration of 1-5ug Affibody® molecule/ml.

References

This product has been referenced in:
  • Petrossian K  et al. ERa-mediated cell cycle progression is an important requisite for CDK4/6 inhibitor response in HR+ breast cancer. Oncotarget 9:27736-27751 (2018). Read more (PubMed: 29963233) »
  • Lallemand C  et al. A Novel System for the Quantification of the ADCC Activity of Therapeutic Antibodies. J Immunol Res 2017:3908289 (2017). Flow Cyt ; Human . Read more (PubMed: 29104875) »
See all 5 Publications for this product

Customer reviews and Q&As

1-6 of 6 Q&A

Question
Answer

I would suggest choosing an ErbB2 negative cell line as your negative control. From the literature, I would suggest the human
neuroblastoma cell line SH-SY5Y which I believe would be a suitable negative control.

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Question
Answer

The ab31891 Anti-ErbB2 Affibody® is not an antibody, but an 'Affibody'. The backbone of Affibody® molecules are derived from a 58 amino acid IgG-binding domain of Staphylococcal Protein A. There is further information on 'Affibodies' on the datasheet for this product. An IgG isotype control works only with antibodies and is not applicable for Affibody® Molecules.

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Answer

Thank you for your inquiry.

I received the following answer from the lab:

"No, the Her2-binder is denoted Z00477, which has similar characteristics, but another sequence than Z00342 (or ZHER2:342, as it also has been called).

See Orlova et al, Cancer Res 2006; 66 4339-48.

Please make the customer aware that ab31891 is a dimer through its peptide backbone (Z-Z-Cys)."

I hope this information is helpful and wish you good luck for your experiments.

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Answer

Thank you for contacting us.

We do not have a FITC-conjugated control for Affibodies®. One palternative would be to conjugate a similar Affibody® to FITC, such as ab31908, which is directed against TNF alpha. The two Affibodies® are similar in structure (head-to-tail dimers) but the suitablility of using an anti-TNF alpha Affibody® as a negative control depends on knowing your samples do not contain TNF alpha.

The online datasheet for the ab31908 is here:

Click here (or use the following: https://www.abcam.com/index.html?datasheet=31908).

The datasheet for our FITC conjugation kit is here:

Click here (or use the following: https://www.abcam.com/index.html?datasheet=102885).

Please do not hesitate to contact us if you need any more advice or information.

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Question

Order Details:

Antibody code:

Ab31891

Batch number:

GR28106-1

Problem (High background, No signal or week signal, Non specific staining):

A very week signal

General Information

Antibody storage conditions (temperature/reconstitution etc):

40C

Description of the problem (high background, low signal, no signal etc.):

The signal was very low and when stain together with other flurofors it was difficult to estimate the results

Sample (Species/Cell type/Cell line etc.):

Human tumor cells growing in SCID mouse

Sample preparation for single cell preparation (Buffer etc.):

The tumor was cut to small piece and was squashed to get single cells in PBS Buffer

Number of cells used:

1 million

Permeabilization, fixation step:

none

Blocking conditions (Buffer/time period, Blocking agent etc.):

none

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):

Ab31891 was used in different concentration from 1 to 20ug as recommended there was no difference in the signal. 45 min incubation washed with PBS+5% FCS


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):

Positive and negative controls used (please specify):

Positive control for the Human cels of the tumor we used APC a CD326, Negative control was Fl Human IgG

Detection method:

Facs

Optimization attempts (problem solving):

How many times have you tried the FACS?

Two times

Have you run a "No Primary" control?

Yes

Do you obtain the same results every time?

Yes

What steps have you altered?

The concentration of the Ab

Additional Notes:

Read More
Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab31891 is tested and covered by our 6 month guarantee for use inflow cytometryand human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimise the results. I would also appreciate if you can confirm some further details:

1. I can recommend to aliquot and store the antibody at -20oC as soon as possible, as directed on the datasheet. this will ensure the antibody remains stable and that we can continue to provide our guarantee.

2. I would appreciate if it is possible to provide some data, including positive and negative controls.

3. I can suggest a permeabilization step would be required in this caseas ErbB2 is localized in the cytoplasm and nucleus. Try permeabilizing in 0.2% Triton for 10 minutes.

4. I would appreciate if you are able to provide any more details of the sample preparation? 'Squashing' the cells in PBS may damage them so that staining is affected, and the cells may not be fully dissociated to single cell suspension. I can recommend to try dissociating the cells in trypsin. There is further information on sample preparation on the following webpage:
http://www.icms.qmul.ac.uk/flowcytometry/uses/multicolouranalysis/cellpreparation/index.html

To ensure the sample is of good quality, a viability cell count in trypan blue can be tried. We recommend there should be over 95% viable cellsin the sample to ensure the sample is healthy enough to proceed with staining and givegood results.

5. This antibody will also detect ErbB2 in the mouse cells. and the CD326 antibody may detect CD326 in the mouse cells. Certainly for this ErbB2 antibody, it has not yet been tested in mouse, and may be cross reacting. It would be useful to includeahuman only positive control, such as SK-BR3 cells?

It would be beneficial to optimize the procedure on a known positive expressing cell line first.

6. What percentage of human Erb2 positive cells are you expecting in the tissue sample?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details including the results of the human positive control..

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Answer

Thank you for contacting us.



Affibody® Molecules are not antibodies, and they do not have different isotypes.

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

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

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