Anti-CXCR4 (phospho S339) antibody (ab74012)

Rabbit polyclonal CXCR4 (phospho S339) antibody. Validated in WB, ELISA, IHC, ICC/IF and tested in Mouse, Human. Cited in 14 publication(s). Independently reviewed in 2 review(s).

Overview

  • Product name

    Anti-CXCR4 (phospho S339) antibody
    See all CXCR4 primary antibodies
  • Description

    Rabbit polyclonal to CXCR4 (phospho S339)
  • Host species

    Rabbit
  • Specificity

    ab74012 detects endogenous levels of CXCR4 only when phosphorylated at serine 339 (Mouse: Ser 346).
  • Tested applications

    Suitable for: WB, ELISA, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic phosphopeptide derived from human CXCR4 around the phosphorylation site of serine 339 (H-S-SP-V-S).

  • Positive control

    • Human brain carcinoma tissue. Extracts from HUVEC cells treated with etoposide (25uM, 24hours). HeLa cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab74012 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 1/500 - 1/1000. Detects a band of approximately 45 kDa (predicted molecular weight: 40 kDa).
ELISA 1/20000.
IHC-P 1/50 - 1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF 1/500 - 1/1000.

Target

  • Function

    Receptor for the C-X-C chemokine CXCL12/SDF-1 that transduces a signal by increasing intracellular calcium ions levels and enhancing MAPK1/MAPK3 activation. Acts as a receptor for extracellular ubiquitin; leading to enhance intracellular calcium ions and reduce cellular cAMP levels. Involved in haematopoiesis and in cardiac ventricular septum formation. Plays also an essential role in vascularization of the gastrointestinal tract, probably by regulating vascular branching and/or remodeling processes in endothelial cells. Could be involved in cerebellar development. In the CNS, could mediate hippocampal-neuron survival. Acts as a coreceptor (CD4 being the primary receptor) for HIV-1 X4 isolates and as a primary receptor for some HIV-2 isolates. Promotes Env-mediated fusion of the virus.
  • Tissue specificity

    Expressed in numerous tissues, such as peripheral blood leukocytes, spleen, thymus, spinal cord, heart, placenta, lung, liver, skeletal muscle, kidney, pancreas, cerebellum, cerebral cortex and medulla (in microglia as well as in astrocytes), brain microvascular, coronary artery and umbilical cord endothelial cells. Isoform 1 is predominant in all tissues tested.
  • Involvement in disease

    Defects in CXCR4 are a cause of WHIM syndrome (WHIM) [MIM:193670]; also known as warts, hypogammaglobulinemia, infections and myelokathexis. WHIM syndrome is an immunodeficiency disease characterized by neutropenia, hypogammaglobulinemia and extensive human papillomavirus (HPV) infection. Despite the peripheral neutropenia, bone marrow aspirates from affected individuals contain abundant mature myeloid cells, a condition termed myelokathexis.
  • Sequence similarities

    Belongs to the G-protein coupled receptor 1 family.
  • Domain

    The amino-terminus is critical for ligand binding. Residues in all four extracellular regions contribute to HIV-1 coreceptor activity.
  • Post-translational
    modifications

    Phosphorylated on agonist stimulation. Rapidly phosphorylated on serine and threonine residues in the C-terminal. Phosphorylation at Ser-324 and Ser-325 leads to recruitment of ITCH, ubiquitination and protein degradation.
    Ubiquitinated by ITCH at the cell membrane on agonist stimulation. The ubiquitin-dependent mechanism, endosomal sorting complex required for transport (ESCRT), then targets CXCR4 for lysosomal degradation. This process is dependent also on prior Ser-/Thr-phosphorylation in the C-terminal of CXCR4. Also binding of ARRB1 to STAM negatively regulates CXCR4 sorting to lysosomes though modulating ubiquitination of SFR5S.
    Sulfation on Tyr-21 is required for efficient binding of CXCL12/SDF-1alpha and promotes its dimerization.
    O- and N-glycosylated. Asn-11 is the principal site of N-glycosylation. There appears to be very little or no glycosylation on Asn-176. N-glycosylation masks coreceptor function in both X4 and R5 laboratory-adapted and primary HIV-1 strains through inhibiting interaction with their Env glycoproteins. The O-glycosylation chondroitin sulfate attachment does not affect interaction with CXCL12/SDF-1alpha nor its coreceptor activity.
  • Cellular localization

    Cell membrane. In unstimulated cells, diffuse pattern on plasma membrane. On agonist stimulation, colocalizes with ITCH at the plasma membrane where it becomes ubiquitinated.
  • Information by UniProt
  • Database links

  • Alternative names

    • C-X-C chemokine receptor type 4 antibody
    • CD184 antibody
    • CD184 antigen antibody
    • Chemokine (C X C motif) receptor 4 antibody
    • Chemokine CXC Motif Receptor 4 antibody
    • CXC-R4 antibody
    • CXCR-4 antibody
    • CXCR4 antibody
    • CXCR4_HUMAN antibody
    • D2S201E antibody
    • FB22 antibody
    • Fusin antibody
    • HM89 antibody
    • HSY3RR antibody
    • LAP 3 antibody
    • LAP3 antibody
    • LCR1 antibody
    • LESTR antibody
    • Leukocyte derived seven transmembrane domain receptor antibody
    • Leukocyte-derived seven transmembrane domain receptor antibody
    • Lipopolysaccharide associated protein 3 antibody
    • Neuropeptide Y receptor Y3 antibody
    • NPY3R antibody
    • NPYR antibody
    • NPYRL antibody
    • NPYY3 antibody
    • NPYY3R antibody
    • Probable G protein coupled receptor lcr1 homolog antibody
    • SDF 1 receptor antibody
    • SDF-1 receptor antibody
    • SEVEN-TRANSMEMBRANE-SEGMENT RECEPTOR antibody
    • Stromal cell derived factor 1 receptor antibody
    • Stromal cell-derived factor 1 receptor antibody
    • WHIM antibody
    • WHIMS antibody
    see all

Images

  • All lanes : Anti-CXCR4 (phospho S339) antibody (ab74012) at 1/500 dilution

    Lane 1 : Extracts from HUVEC cells
    treated with etoposide (25uM, 24hours)
    Lane 2 : Extracts from HUVEC cells
    treated with etoposide (25uM, 24hours) with the immunising phosphopeptide at 5 µg

    Lysates/proteins at 5 µg per lane.

    Predicted band size: 40 kDa
    Observed band size: 45 kDa
    why is the actual band size different from the predicted?

  • Immunohistochemistry analysis of paraffin-embedded human brain carcinoma tissue using ab74012, at 1/50 dilution, in the presence (right panel) or absence (left panel) of the immunising phosphopeptide.
  • Immunofluorescence analysis of HeLa cells using ab74012, at 1/500 dilution, in the presence (right panel) or absence (left panel) of the immunising phosphopeptide.

References

This product has been referenced in:

  • Chen Y  et al. CUDC-907 blocks multiple pro-survival signals and abrogates microenvironment protection in CLL. J Cell Mol Med 23:340-348 (2019). Read more (PubMed: 30353642) »
  • Cornelison RC  et al. Convective forces increase CXCR4-dependent glioblastoma cell invasion in GL261 murine model. Sci Rep 8:17057 (2018). Read more (PubMed: 30451884) »
See all 15 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (10% SDS PAGE)
Sample
Mouse Tissue lysate - whole (Liver)
Specification
Liver
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jul 30 2014

Question
Answer

DISCOUNT CODE: xxx
Expiration date: xxx

I am very pleased to hear you would like to accept our offer and test ab74012 in chicken. This code will give you: 1 free PRIMARY ANTIBODYbefore the expiration date. To redeem this offer, please submit an Abreview for chickenand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount.

Read More

Answer

Thank you for your inquiry.

I can confirm that both antibodies are tested and guaranteed for mouse and IHC-P.

They do bind to different epitopes in the CXCR4 protein and ab74012 will only bind to the CXCR4 protein when it is phosphorylated at serin339.

I hope this information is helpful and wish you and your customer a nice weekend.

Read More

Question

Dear ,

Please find the completed sheet.
1) Abcam product code ab
ab74012
2) Abcam order reference number or product batch number
Lot:GR51025-1
3) Description of the problem
No specific band was detected
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysate (treated with SDF-1 or pervanadate vs untreated, cells have been starved or not starved)
Lysis buffer : RIPA
50 mM TrisHCl pH7.4; 150 mM NaCl; 2 mM EDTA; 1% NP-40; 0.1%SDS
1 liter:
1 M tris 7.4 (invitrogen #15567-027) 50 ml
5M NaCl 30 ml
0.5M EDTA (Gibco #15575-038) 4 ml
NP40 10 ml
10%SDS (Bio-rad #161-0416) 10 ml
Add the following inhibitors just prior use:
PIC (protease inhibitor cocktail):100x Sigma #P8340
DTT 1M : 1000X (1mM final) Sigma #646563
NaF 0.5M: 100X (5mM final) Fluka # 67414
Na3VO4 200mM: 100X(2mM final) Sigma #S6508
Protease inhibitors: see above
Phosphatase inhibitors : see above
Reducing agent: see above
Boiling for ≥5 min? yes and no (95°C and 37°C)
Protein loaded ug/lane or cells/lane: 10µg
Positive control : 100nM SDF-1, 50µM pervadate
Negative control : water
5) Percentage of gel
Type of membrane : Immobilon-P transfer membrane, 0.45µm Millipore #IPVH00010
Protein transfer verified: yes
Blocking agent and concentration: milk or BSA, 5% each in PBST or TBST
Blocking time : 2h at 4°C
Blocking temperature: 2h at 4°C
6) Was a fresh membrane used or had it been probed and stripped with a different antibody?
Fresh membrane used
7) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution : 1:500
Diluent buffer: 5%BSA/PBST, 5%BSA/TBST, 5%milk/PBST, 5%milk/TBST
Incubation time: overnight at 4°C
Incubation temperature: overnight at 4°C
8) Secondary antibody:
Species: donkey anti-rabbit HRP Jackson Immuno Research #705-035-152
Reacts against:
Concentration or dilution : 1:5000
Diluent buffer: 5%BSA/PBST, 5%BSA/TBST, 5%milk/PBST, 5%milk/TBST
Incubation time 30min at 4°C
Incubation temperature: 30min at 4°C
Fluorochrome or enzyme conjugate: HRP
9) Washing after primary and secondary antibodies:
Buffer: PBST or TBST
Number of washes: 3 times
10)Detection method: chemiluminescence, Immobilon Western Millipore #WBKLS0500
11) How many times have you run this staining? 3 times
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? Blocking and buffer, heating or none heating of samples before loading
blocked with 5%BSA/PBS
blocked with 5%milk/PBST
Thanks for your answer.
Best Regards

Read More
Answer

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

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab74012. I would also appreciate if you can confirm some further details:

What cell line was used for the western blot? And for how long were they treated with the SDF-1 and pervadate?

The Western blot presented on the datasheet of this antibody was performed using a 1/500 dilution with overnight incubation at 4°Cas you have been doing, however a larger amount of protein was loaded,60 ug. BSA was used for blocking.

I would suggest rying the following:

1. Heating the protein sample to 70°C for 10 minutes prior to loading onto the gel. As the protein is a multi-pass membrane protein heating to 95°C can sometimes lead to aggregation.

2. I would suggest loading more protein onto the gel. Initially try 30 ug per lane.

3. I would block the membrane using 3% BSA instead of milk as milk contains the phosphoprotein casein which can sometimes increase the background observed. I would also increase thetemperature of the blocking step to room temperature for 2 hours.

4. I would incubate the secondary antibody for a little longer, 2 hours at room temperature.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Read More

Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with the anti-CXCR4 (phospho S339) antibody (ab74012). We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. I have contacted the source of this antibody to obtain the exact protocol used to produce the Western blot image presented on the datasheet of ab74012.

In the meantime, I may be able to help in optimising the protocol if you wouldn't mind sharing it with me? This will also allow me to investigate this problem further and initiate any additional testing where necessary. To this end I am attachinga questionnaire. If you could also include an image of the results obtained that would be very helpful to further understand the problems encountered.

If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

Read More

Answer

Thank you for your response and for providing some further details.

I would like to clarify a few points in this e-mail.

This (ab74012: Anti-CXCR4 (phospho S339)) is a polyclonal antibody (not a monoclonal) and will therefore consist of a mixed population of IgGs each of which will recognize a different epitope. For polyclonal antibodies, the epitope sequence is not mapped. Such studies can only be carried out for monoclonal antibodies. The epitope of ab74012 is unknown since it has not been determined.

The exact sequence of the immunogen used to raise this antibody is commercially sensitive information so it is not provided on the on-line datasheet. It was a synthetic phosphopeptide derived from human CXCR4 around the phosphorylation site of serine 339 (H-S-SP-V-S).

The specificity of this antibody is QC-d on a regular base.

1. The immunogen peptide must be HPLC purified and MS to confirm the sequence is correct.

2. The antibody binding can be blocked specifically with the immunogen peptide.

3. For phospho-specific antibody, ELISA data must show that this antibody react with phosphor-peptide, not the counterpart (non-phospho-peptide with the same amino acid sequence).

I have searched the immunogen sequence using the database from UniProt and didn’t find any significant homology with other proteins. Could you please specify and confirm what blast website you used and what results were.

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. We have been selling this antibody since 2009. According to our record, this is the first complaint we have received so not aware of any specificity issues.

As recommended in the previous e-mail, blocking phospho protein properly is essential. For detection of phospho protein, we would suggest using 1-3% BSA solution - as blocking. Some other types of blocking agents may lead to significant decrease or loss of signal intensity due to reaction of primary antibody with blocking/diluting agent.

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

Thank you for getting back to me and for providing some further details.

This antibody detects endogenous levels of CXCR4 only when phosphorylated at serine 339 (Mouse: Ser 346).

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) Do you know if the samples contain phosphorylated CXCR4? Have you had any chance to use freshly fixed samples?

2) For detection of phospho protein, we would suggest using 1-3% BSA solution - as blocking. Some other types of blocking agents may lead to significant decrease or loss of signal intensity due to reaction of primary antibody with blocking/diluting agent.

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Read More

Answer

Thank you for your enquiry regarding ab74012 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.

The immunogen used to raise this antibody was a synthetic phosphopeptide derived from human CXCR4 around the phosphorylation site of serine 339 (H-S-SP-V-S). Unfortunately, this peptide is not commercially available.

Could you provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.

I am particularly interested in the followings:

- sample preparation,

- blocking agent and time,

- positive control,

- secondary (no primary) control etc

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
Application
Western blot
Sample
Rat Cell lysate - whole cell (Mesenchymal stem cell)
Loading amount
15 µg
Specification
Mesenchymal stem cell
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 18 2009

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