Overview

  • Product name
    Anti-Eph receptor A4 antibody
    See all Eph receptor A4 primary antibodies
  • Description
    Rabbit polyclonal to Eph receptor A4
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, ELISA, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human Eph receptor A4 aa 875-904 (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).

  • Positive control
    • NCI-H460 cell lysate
  • General notes

    Protein kinases are enzymes that transfer a phosphate group from a phosphate donor, generally the g phosphate of ATP, onto an acceptor amino acid in a substrate protein. By this basic mechanism, protein kinases mediate most of the signal transduction in eukaryotic cells, regulating cellular metabolism, transcription, cell cycle progression, cytoskeletal rearrangement and cell movement, apoptosis, and differentiation. With more than 500 gene products, the protein kinase family is one of the largest families of proteins in eukaryotes. The family has been classified in 8 major groups based on sequence comparison of their tyrosine (PTK) or serine/threonine (STK) kinase catalytic domains. The tyrosine kinase (TK) group is mainly involved in the regulation of cell-cell interactions such as differentiation, adhesion, motility and death. There are currently about 90 TK genes sequenced, 58 are of receptor protein TK (e.g. EGFR, EPH, FGFR, PDGFR, TRK, and VEGFR families), and 32 of cytosolic TK (e.g. ABL, FAK, JAK, and SRC families).

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer
    Preservative: 0.09% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    Protein G purified
  • Purification notes
    This antibody is purified through a protein G column and eluted out with both high and low pH buffers and neutralized immediately after elution then followed by dialysis against PBS.
  • Primary antibody notes
    Protein kinases are enzymes that transfer a phosphate group from a phosphate donor, generally the g phosphate of ATP, onto an acceptor amino acid in a substrate protein. By this basic mechanism, protein kinases mediate most of the signal transduction in eukaryotic cells, regulating cellular metabolism, transcription, cell cycle progression, cytoskeletal rearrangement and cell movement, apoptosis, and differentiation. With more than 500 gene products, the protein kinase family is one of the largest families of proteins in eukaryotes. The family has been classified in 8 major groups based on sequence comparison of their tyrosine (PTK) or serine/threonine (STK) kinase catalytic domains. The tyrosine kinase (TK) group is mainly involved in the regulation of cell-cell interactions such as differentiation, adhesion, motility and death. There are currently about 90 TK genes sequenced, 58 are of receptor protein TK (e.g. EGFR, EPH, FGFR, PDGFR, TRK, and VEGFR families), and 32 of cytosolic TK (e.g. ABL, FAK, JAK, and SRC families).
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab5396 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
ICC/IF 1/500.
ELISA 1/1000.
WB 1/1000. Detects a band of approximately 108 kDa (predicted molecular weight: 118 kDa).

We recommend using CHAPS lysis buffer rather than RIPA, Invitrogen NuPage gel system, blotting on PVDF membrane, blocking 30' in 10% Fetal Bovine Serum, 3% BSA, 0.3% Tween 20 in 1X PBS - please see Abreview for further information (published on 24th July, 2006).

IHC-P 1/25 - 1/100.

Target

  • Function
    Receptor for members of the ephrin-A family. Binds to ephrin-A1, -A4 and -A5. Binds more poorly to ephrin-A2 and -A3. May play a role in a signal transduction process involved in hindbrain pattern formation.
  • Tissue specificity
    Ubiquitous.
  • Sequence similarities
    Belongs to the protein kinase superfamily. Tyr protein kinase family. Ephrin receptor subfamily.
    Contains 2 fibronectin type-III domains.
    Contains 1 protein kinase domain.
    Contains 1 SAM (sterile alpha motif) domain.
  • Domain
    The protein kinase domain mediates interaction with NGEF/ephexin-1.
  • Cellular localization
    Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • Cek 8 antibody
    • CEK8 antibody
    • EK8 antibody
    • eph receptor a4 antibody
    • EPH-like kinase 8 antibody
    • EPHA4 antibody
    • EPHA4_HUMAN antibody
    • Ephrin type-A receptor 4 antibody
    • HEK 8 antibody
    • hEK8 antibody
    • Receptor protein-tyrosine kinase HEK8 antibody
    • Sek 1 antibody
    • SEK antibody
    • TYRO 1 protein tyrosine kinase antibody
    • TYRO1 antibody
    • Tyrosine-protein kinase receptor SEK antibody
    • Tyrosine-protein kinase TYRO1 antibody
    see all

Images

  • All lanes : Anti-Eph receptor A4 antibody (ab5396) at 1/1000 dilution

    Lane 1 : HeLa Cell Lysate
    Lane 2 : NCI-H460 Cell Lysate
    Lane 3 : Mouse NIH3T3 Cell Lysate
    Lane 4 : Human ovary tissue Lysate

    Lysates/proteins at 35 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit IgG H+L HRP conjugated at 1/10000 dilution

    Predicted band size: 118 kDa



    Incubation time was overnight at 4°C. Blocking/Dilution buffer: 5% NFDM/TBST.

  • Anti-Eph receptor A4 antibody (ab5396) at 1/1000 dilution + NCI-H460 whole cell lysate at 35 µg

    Secondary
    Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/10000 dilution

    Predicted band size: 118 kDa



    Blocking/Dilution buffer: 5% NFDM/TBST.

  • ab5396 at 1/500 staining rat cerebellar granual cells by ICC/IF. The cells were permeabilized with 0.1% Triton X-100, paraformaldehyde fixed and blocked with 2% serum prior to incubation with the antibody for 1 hour. A Cy3 conjugated goat anti-rabbit was used as the secondary.

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labeling Eph receptor A4 with ab5396 at dilution 1/25. Tissue was fixed with formaldehyde and blocked with 3% BSA for 0.5 hour at 37°C; heat mediated antigen retrieval was performed using a citrate buffer (pH6). Samples were incubated with primary antibody (1/25) for 1 hour at 37°C. A Peroxidase-conjugated Goat anti-rabbit polyclonal (ready to use) was used as the secondary antibody.

References

This product has been referenced in:
  • Feng L  et al. EphA4 may contribute to microvessel remodeling in the hippocampal CA1 and CA3 areas in a mouse model of temporal lobe epilepsy. Mol Med Rep 15:37-46 (2017). Read more (PubMed: 27959424) »
  • Todd KL  et al. EphA4 Regulates Neuroblast and Astrocyte Organization in a Neurogenic Niche. J Neurosci 37:3331-3341 (2017). ICC/IF ; Mouse . Read more (PubMed: 28258169) »
See all 3 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Abreviews
Abcam has not validated the combination of species/application used in this Abreview.
Application
IHC - Wholemount
Sample
Marmoset (common) Tissue (Adult Brain)
Specification
Adult Brain

Dr. 煜欽 林

Verified customer

Submitted Jun 05 2013

Question
Answer

The new lot has been tested in WB so it will be OK.

I have requested image from laboratory, which I forward to you soon.

Read More

Answer

Thank you very much for having patience.

We have now acquired new lot of this antibody. I will inform you soon with the shipment date.

Please let me know if you have any question.

Read More

Answer

Thank you for your email.

I have been in contact with laboratory for acquiring new lot. I will send you a confirmation once we ship it.

Many thanks for having patience. Please do not hesitate to contact me if you have any question.

Read More

Question
Answer

Dr. Tanya is on holiday until 2nd January so I am dealing with her inquires.

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

I apologize for the inconvenience and am pleased to offer you a free of charge replacement from different lot.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

PS: Please provide the purchase order number which will help us to send you the replacement at same address if you are interested in trying a different lot.

Read More

Question

Hi,



Here is an image of IHC and western blot for EPHA4 and some information on the IHC protocol.



IHC: We often obtain nuclear staining. Have you found that also?



Western: The size is approx 90-95 (three different cell lines) but the predicted size is 108kD.



1) Abcam product code ab 5396



2) Abcam order reference number or product batch number GR223521



3) Description of the problem attached IHC image. We often observe nuclear staining. Have you experienced that?



4) Sample preparation:

Species

Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other:

Sample preparation

Positive control

Negative control



5) Fixation step

Yes/No

If yes: Fixative agent and concentration

Fixation time

Fixation temperature



6) Antigen retrieval method reveal in in a decloaking chamber according to the manufacturer’s instructions (Biocare, Concord, CA, USA)



7) Permeabilization method:

Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?

Permeabilizing agent and concentration:





8) Blocking agent (eg BSA, serum…): 2.5 % horse serum

Concentration

Blocking time 20 min

Blocking temperature room temperature



9) Endogenous peroxidases blocked?yes

Endogenous biotins blocked?



10) Primary antibody (If more than one was used, describe in “additional notes”) :EPHA4

Concentration or dilution 1:100

Diluent buffer

Incubation time



11) Secondary antibody: incubation using streptavidin/peroxidase complex was performed according to the manufacturer’s manual (Vectastain Universal Quick Kit, Vector Laboratories Inc., Burlingame, CA, USA)

Species:

Reacts against:

Concentration or dilution

Diluent buffer

Incubation time

Fluorochrome or enzyme conjugate



12) Washing after primary and secondary antibodies:yes

Buffer TBS

Number of washes 3



13) Detection method incubation using streptavidin/peroxidase complex was performed according to the manufacturer’s manual (Vectastain Universal Quick Kit, Vector Laboratories Inc., Burlingame, CA, and development step with 3,3’́-diaminobenzidine



14) How many times have you run this staining?

Do you obtain the same results every time?

What steps have you altered to try and optimize the use of this antibody?



Document attachment: Attaching images of your IHC is strongly recommended and can greatly speed up our investigation of your problem.





I hope that you can help to clarify this.



Regards,

Read More
Answer

Thank you for your enquiry regarding ab5396 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem. I need to deal with the 2 applications separately and appreciate some further details:

IMMUNOSTAINING:

1) Sample preparation: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other

Question: Could you please specify the species, cell or tissue types used? This information is crucial for us for troubleshooting and it is not provided in this and your previous e-mail.

2) If yes: Fixative agent and concentration

Question: What exactly used for fixing the sample? Could you please confirm? Aldehyde or alkohol-based?

3) Antigen retrieval method reveal in in a decloaking chamber according to the manufacturer’s instructions (Biocare, Concord, CA, USA)

Question: Did you apply heat or enzyme-based antigen retrial and any optimization steps were performed?

4) Blocking agent (eg BSA, serum…): 2.5 % horse serum, Blocking time 20 min

Question: Have you tried 10% serum or 5% BSA for 1 hr at RT?

WESTERN BLOT:

Questions:

- What lysis buffer was used for sample preparation i.e. any proteinsea inhibitors included?

- How the non-specific bindings were blocked?

- How much protein was loaded?

- Has the detection system been checked or no primary antibody run?

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Read More

Answer

I am sorry to hear that you have been experiencing problems using this product in the application that you wish.

I can confirm that ab5396 has been tested for both Western blot and for Immunohistochemistry.

In order to assess the quality of our products I would ask that you complete a brief questionnaire relating to the application used. Often it is possible to make suggestions that may help resolve problems experienced using a particular product.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience.

Could you please provide some further details of the protocol used and complete the following forms WB and IHC (attached as word documents). It would be much appreciated if you could attach an image to the response.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - whole (Brain C57Bl/6 mice)
Loading amount
30 µg
Specification
Brain C57Bl/6 mice
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 3%

Abcam user community

Verified customer

Submitted Aug 23 2006

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (P0 RAT Spinal Cord)
Loading amount
30 µg
Specification
P0 RAT Spinal Cord
Blocking step
Other as blocking agent for 30 minute(s) · Concentration: 3%BSA, 10%

Abcam user community

Verified customer

Submitted Aug 14 2006

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Cerebellar granule cells)
Specification
Cerebellar granule cells
Fixative
Paraformaldehyde
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 2% goat se

Abcam user community

Verified customer

Submitted Aug 09 2006

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

Sign up