Anti-DLL4 antibody (ab7280)

Rabbit polyclonal DLL4 antibody. Validated in WB, ELISA, IHC, ICC/IF and tested in Mouse, Human. Cited in 60 publication(s). Independently reviewed in 8 review(s).

Overview

  • Product name

  • Description

    Rabbit polyclonal to DLL4
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, WB, ICC/IF, ELISAmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide:

    AWHAPGDDLRPEAL-C

    conjugated to KLH, corresponding to amino acids 121-134 of Human Delta4.

  • Positive control

    • Recombinant human DLL4 protein (ab84081)

Properties

Applications

Our Abpromise guarantee covers the use of ab7280 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 1/50.
WB 1/500 - 1/2500. Detects a band of approximately 74 kDa (predicted molecular weight: 74 kDa).

The levels of DLL4 in endogenous samples may be low. Abcam has not been successful in generating western blot data using endogenous samples, but can demonstrate the specificity using recombinant proteins and overexpressing cell lysates. Some customers have been successful using ab7280 for these samples - please see published references below.

ICC/IF Use at an assay dependent concentration.
ELISA 1/10000 - 1/50000.

Target

  • Function

    Plays a role in the Notch signaling pathway. Activates Notch-1 and Notch-4.
  • Tissue specificity

    Expressed in vascular endothelium.
  • Sequence similarities

    Contains 1 DSL domain.
    Contains 8 EGF-like domains.
  • Domain

    The Delta-Serrate-Lag2 (DSL) domain is required for binding to the Notch receptor.
  • Post-translational
    modifications

    Ubiquitinated by MIB (MIB1 or MIB2), leading to its endocytosis and subsequent degradation.
  • Cellular localization

    Membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • AOS6 antibody
    • Delta 4 antibody
    • delta 4 precursor antibody
    • Delta ligand 4 antibody
    • delta ligand 4 precursor antibody
    • Delta like 4 antibody
    • Delta like 4 homolog antibody
    • Delta like 4 protein antibody
    • Delta like canonical Notch ligand 4 antibody
    • Delta like protein 4 antibody
    • Delta-like 4 (Drosophila) antibody
    • Delta-like protein 4 antibody
    • Delta4 antibody
    • DLL 4 antibody
    • Dll4 antibody
    • DLL4_HUMAN antibody
    • Drosophila Delta homolog 4 antibody
    • hdelta2 antibody
    • Homeobox protein DLL-4 antibody
    • MGC126344 antibody
    • Notch ligand delta 2 antibody
    • Notch ligand DLL4 antibody
    • Notch ligand DLL4 precursor antibody
    • XDLL-4 antibody
    see all

Images

  • All lanes : Anti-DLL4 antibody (ab7280) at 1/1000 dilution

    Lane 1 : Recombinant human DLL4 at 0.05 µg
    Lane 2 : Whole cell lysate from HEK293 over-expressing human DLL4. at 10 µg
    Lane 3 : Whole cell lysate from control HEK293 . at 10 µg

    Secondary
    All lanes : Peroxidase labelled anti-rabbit antibody at 1/40000 dilution

    Performed under reducing conditions.

    Predicted band size: 74 kDa



    This blot was produced under denaturing, reducing conditions. After transfer, the membrane was blocked for half an hour before being incubated overnight at 4°C with rabbit polyclonal to DLL4 (ab7280; diluted 1:1000).  Antibody binding was detected using peroxidase labelled anti-rabbit antibody (diluted 1:40,000) for half an hour at room temperature and visualised using ECL development solution.

  • IHC image of ab7280 staining in human kidney formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab7280, 5µg/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.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ICC/IF image of ab7280 stained Hek293 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7280, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:

  • Zhang Y  et al. MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD. Am J Physiol Lung Cell Mol Physiol 316:L144-L156 (2019). Read more (PubMed: 30382766) »
  • Pattanaik S  et al. Vascular Tissue Engineering Using Scaffold-Free Prevascular Endothelial-Fibroblast Constructs. Biores Open Access 8:1-15 (2019). Read more (PubMed: 30637179) »
See all 65 Publications for this product

Customer reviews and Q&As

1-10 of 18 Q&A

Answer

Thank you very much for your cooperation so far and sending me the additional information.

I have one last tip i would like to share with you:

I would like to suggest to either decrease the H2O2 percentage down to 0.3% and to incubate for 10 minutes, or to block the endogenous peroxidase after the primary antibody incubation to avoid possible epitope alteration.

Please let me know if my tips improved the results.

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Answer

Je peux vous confirmer que 0.2% Tween a été utilisé pour les immunoblots et que le lysat de pancréas de souris a été chauffé avant dépôt sur le gel de polyacrylamide.

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Answer


Unfortunately it is not possible for us to sell the pre-immune serum of our rabbits.
I am sorry that on this occasion I could not be of more help.

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Answer

Is the result as before: a strong band at the expected molecular weight of DLL4, and another smaller band at 72 kDa?

Can you tell me if the customer has tried any other DLL4 antibodies with these samples? Also, have they considered isolating a membrane fraction from the cell lysate? That would enrich mature DLL4 in the sample and might eliminate the 72 kDa band, if it is not a membrane-bound protein like DLL4.

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Answer

Thank you for contacting us.
Both of these antibodies are shipped at 4C on blue ice packs. They are both in stock, so if you order today by 8pm within the US, you will receive them by 10am tomorrow.
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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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry these products did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 123456.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Answer

Thank you very much for your reply and your cooperation so far.

I have found your order for these two products, and would be happy to send you a replacement. Please let me know which products you have chosen from our catalog.

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Answer

Thank you for contacting us again.

I am sorry to hear that the protocol tips did not improve the results. Please let me know when you have ordered the two antibodies. In addition, did you buy them directly from us or via our distributor?


I am looking forward to your reply.

Read More

Question

Good morning,

this is the completed questionnaire. There are also two images.

Thank you for your time and cooperation



1) Abcam product code ab: Lgr5 (rabbit policlonal anti-GPR49 ab75732) and Dll-4 (rabbit policlonal anti-DLL4 Ab-7280)

2) Abcam order reference number or product batch number

3) Description of the problem : We have tested these antibodies using different protocols with no results

4) Sample preparation
Species: mouse
Type of sample: formalin fixed paraffin embedded sections
Sample preparation
Positive control
Negative control

5) Fixation step
Yes
If yes: Fixative agent and concentration : formalin 4%
Fixation time: over night
Fixation temperature: 4°C

6) Antigen retrieval method : antigen retrieval was performed by autoclave with DIVA 3’ at 125°C and 10’ at 90°C, by boiling sections for 10’ in 10mM sodium citrate buffer (pH 6) in a microwave oven

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…): RODENT BLOCK M and BACKGROUND SNIPER
Concentration: ready for use
Blocking time: 20’
Blocking temperature : room temperature

9) Endogenous peroxidases blocked? Yes, with PEROXIDASED at room temperature
Endogenous biotins blocked?

10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution: for LGR5 1:25,1:50 and 1:200, for DLL-4 1:50, 1:200, 1:500,1:1000
Diluent buffer : DA VINCI GREEN
Incubation time: 60’ room temperature

11) Secondary antibody: MACH4 HRP POLYMER
Reacts against: rabbit
Concentration or dilution : ready for use
Diluent buffer
Incubation time: 30’
Fluorochrome or enzyme conjugate

12) Washing after primary and secondary antibodies:
Buffer: PBS 1x with 0.05% tween
Number of washes: two times

13) Detection method: DAB

14) How many times have you run this staining? Three times
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? Antigen retrieval , antibody dilution
Document attachment: Attaching images of your IHC is strongly recommended and can greatly speed up our investigation of your problem.

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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 appreciate if you can confirm some further details. In addition, I would like to make some suggestions:

1. Have you fixed thetissue prior to embedding?

2. What kind of tissue are you testing?

3. How high is the concentration of the H2O2 and for how long have you applied this? Was the peroxidase blocking donebefore or after the primary antibody incubation? I would like to suggestusing aH2O2 percentageof 0.3% and to incubate for 10 minutes, or to block the endogenous peroxidase after the primary antibody incubation to avoid possible epitope alteration.

4. Using the autoclave is quite a harsh method, whereas boiling for 10 min via microwave may have not been efficient enough. I would like to suggest to stick to the microwave method, but to perform a time series of 15, 20 and 25 min to find the appropriate retrieval point.

5. For both antibodies I would suggest increasing the incubation time to overnight at 4C. For ab75732 increase concentration to 1/10 as suggested on the datasheet.

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.

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Answer

Thank you for contacting us. I would recommend ab10554 (Dll1) and ab7280 (abDll4), which are both guaranteed for ICC im mouse. We have not tested these products for mouse intestinal staining specifically but do guarantee their use in mouse under our Abpromise. This means within 6 months of purchase we can replace, refund or credit you for this product if it is not satisfactory.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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1-10 of 18 Q&A

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