Overview

  • Product name

  • Description

    Rabbit polyclonal to SPRED1
  • Host species

    Rabbit
  • Specificity

    ab77079 is predicted to have no cross-reactivity to SPRED2 or SPRED3.
  • Tested applications

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

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic 14 amino acid peptide from near the center of human SPRED1 (NP_689807).

  • Positive control

    • Human brain tissue lysate. This antibody gave a positive result in IF in the following Formaldehyde fixed cell line: A549.

Properties

Applications

Our Abpromise guarantee covers the use of ab77079 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 - 2 µg/ml. Predicted molecular weight: 50 kDa.
ICC/IF Use a concentration of 5 µg/ml.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

  • Function

    Tyrosine kinase substrate that inhibits growth-factor-mediated activation of MAP kinase. Negatively regulates hematopoiesis of bone marrow.
  • Tissue specificity

    Weakly expressed in embryonic cell line (HEK-293).
  • Involvement in disease

    Defects in SPRED1 are the cause of Legius syndrome (LEGIUSS) [MIM:611431]. It is a disorder characterized mainly by cafe au lait macules without neurofibromas or other tumor manifestations of neurofibromatosis type 1, axillary freckling, and macrocephaly. Additional clinical manifestations include Noonan-like facial dysmorphism, lipomas, learning disabilities and attention deficit-hyperactivity.
  • Sequence similarities

    Contains 1 KBD domain.
    Contains 1 SPR (sprouty) domain.
    Contains 1 WH1 domain.
  • Post-translational
    modifications

    Phosphorylated on tyrosine.
  • Cellular localization

    Cell membrane. Membrane > caveola. Nucleus. Localized in cholesterol-rich membrane raft/caveola fractions.
  • Information by UniProt
  • Database links

  • Alternative names

    • EVH1 domain-containing protein 1 antibody
    • EVH1/Sprouty domain containing protein antibody
    • FLJ33903 antibody
    • hSpred 1 antibody
    • hSpred1 antibody
    • NFLS antibody
    • PPP1R147 antibody
    • protein phosphatase 1 regulatory subunit 147 antibody
    • SPRE1_HUMAN antibody
    • SPRED 1 antibody
    • Spred-1 antibody
    • spred1 antibody
    • Sprouty related EVH1 domain containing 1 antibody
    • sprouty related EVH1 domain containing protein 1 antibody
    • Sprouty related protein 1 with EVH 1 domain antibody
    • Sprouty-related antibody
    • Suppressor of Ras/MAPK activation antibody
    see all

Images

  • Lane 1 : Anti-SPRED1 antibody (ab77079) at 1 µg/ml
    Lane 2 : Anti-SPRED1 antibody (ab77079) at 2 µg/ml

    All lanes : Human brain tissue lysate

    Lysates/proteins at 15 µg per lane.

    Predicted band size: 50 kDa
    Observed band size: 50 kDa

  • IHC image of ab77079 staining in human cerebral cortex 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 ab77079, 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.

    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.

  • ab77079 stained A549 cells. The cells were 4% formaldehyde 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 ab77079 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.

  • Immunofluorescence of Spred1 in Human Brain cells using ab77079 at 20 ug/ml.

References

This product has been referenced in:

  • Susanto A  et al. Spred negatively regulates lens growth by modulating epithelial cell proliferation and fiber differentiation. Exp Eye Res 178:160-175 (2019). Read more (PubMed: 30290165) »
  • Wang L  et al. miR-126 Regulation of Angiogenesis in Age-Related Macular Degeneration in CNV Mouse Model. Int J Mol Sci 17:N/A (2016). WB ; Mouse . Read more (PubMed: 27338342) »
See all 5 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Question
Answer

Thank you very much for your reply.
I'm sending a free of charge replacement vial of ab64740 on the order *** which should arrive tomorrow.
Please keep me updated about the performance of this antibody, and let me know if there is anything else that we can do for you. Have a nice day.

Read More

Answer

Thank you very much for completing our recent survey about your experience with us.

I am sorry the replacement SPRED1 antibody is also not working in WB. With ab64740, are you still observing the band ˜85 kDa, or are you not detecting any signal? If you are not getting any signal, it may help to try a primary antibody concentration of 2ug/mL with a secondary antibody dilution of 1:2000-5000.

If these suggestions are not helpful, I can offer you a refund, credit, or a replacement with a different antibody if you prefer. Please let me know how you would like to resolve this issue, and I'll be happy to help.

Have a nice day.

Read More

Answer

Thank you very much for keeping me updated with the results.
I'm sorry to see that the suggestions were not more helpful. I'd be happy to send a different SPRED1 antibody to try. We also have ab64740 and ab62911-
https://www.abcam.com/SPRED1-antibody-M23-P2G3-ab64740.html
https://www.abcam.com/SPRED1-antibody-ab62911.html
Would you like to try one of these as a replacement? If so, please send me your original order or PO number and I will set this up promptly.
Please let me know if you have any additional questions or comments, or if there is anything else that we can do for you. Have a nice day.

Read More

Answer

Thank you for your reply. I am sorry the replacement is also not working in WB. With ab64740, are you still observing the band ˜85 kDa, or are you not detecting any signal? It would be very helpful if you could please provide an image of your results. If you are not getting any signal, it may help to try a primary antibody concentration of 2ug/mL with a secondary antibody dilution of 1:2000-5000. I hope this helps, if not, please let me know and I will be happy to offer you a refund or credit for your original purchase.

Read More

Question
Answer

Thank you very much for your call today and for letting us know about the trouble with this antibody.

As we discussed, I'm sending a free of chargevial of ab64740 on the order ***, which should arrive tomorrow. Please keep me updated about the results with this replacement antibody.

I look forward to hearing from you. Please let me know if you have any questions or if there is anything else that we can do for you.

Read More

Question
Answer

Thank you very much for contacting us and letting us know about the trouble with this antibody.
To improve the results, I would suggest using 5% milk to block the membrane, and also blocking overnight at 4C. You an additionally load less than 40 ug of protein to reduce the non-specific binding, around 20 ug will be sufficient.
If these results do not improve the results, we do fully guarantee the results of this antibody in Western blot with your samples, so I will be happy to send a replacement antibody or issue a credit or refund if you prefer.
Please let me know if you have any questions or if there is anything else that we can do for you, and I'll be happy to help.

Read More

Question
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 provide us with vital information for our monitoring of product quality. I would like to reassure you that this antibody is tested and covered by our guarantee for WB and human samples.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.

I can recommend you may like to consider trying the following options:

1. Try more diluted antibody 1:2000 or even 1:3000. This should help to reduce non specific staining.

2. Has the quality of the sample been assessed using a loading control? Try some fresh samples.

Alternatively, or if these tips do not work, I am pleased to offer you a free of charge replacement or credit note in compensation.

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

Read More

Answer

Thank you very much for contacting us and letting us know about the trouble with this antibody.

The details and image you provided are very helpful. Could you also tell me how the samples were prepared prior to electrophoresis? How long were they boiled and in what kind of sample buffer? The SPRED1 protein can dimerize with itself and with other proteins, so I'd recommend fully reducing the samples to make sure no dimers are present.

I've looked through the protocol used to test this antibody in Western blot, and the lab used a 5% skim milk solution for blocking and antibody dilution, so it may be helpful to use milk instead of BSA. You can also block the membrane overnight at 4C, and only incubate with the primary antibody for 1 hour at room temperature. These steps can help reduce the non-specific binding. If you do decide to try these suggestions, please keep me updated about any future results.

We do fully guarantee this antibody to work in Western blot with mouse lysates, so if the results are not satisfactory then we can send a replacement or alternatively issue a credit or refund. Please let me know if you would prefer one of these options.

I look forward to hearing from you. If there is anything else that we can do for you, please let me know and I'll be happy to help.

Read More

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