Overview

  • Product name
  • Description
    Chicken polyclonal to SPON1
  • Host species
    Chicken
  • Tested applications
    Suitable for: WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Immunogen

    Synthetic peptide, corresponding to amino acids 549-807 of SPON1.

Properties

Applications

Our Abpromise guarantee covers the use of ab14271 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 1/2000. Predicted molecular weight: 100.9 kDa.
IHC-P Use at an assay dependent concentration. PubMed: 24294399

Target

  • Relevance
    SPON1 is a member of a subgroup of the thrombospondin type 1 (TSR) class molecules, defined by two domains of homology, the FS1/FS2 and TSR domains. The TSRs of SPON1 proteins are typical of class 2 TSRs. SPON1, which is similar to thrombospondin, is a extracellular matrix attached molecule that promotes neurite outgrowth and inhibits angiogenesis. Analysis of gain and loss of function experiments reveal that SPON1 is required for accurate pathfinding of embryonic axons, and plays a dual role in patterning axonal trajectories. It promotes the outgrowth of commissural and inhibits the outgrowth of motor axons, and has also been implicated in inflammatory processes in the nervous system.
  • Cellular localization
    Secreted protein; extracellular space; extracellular matrix.
  • Database links
  • Alternative names
    • F spondin antibody
    • SPON 1 antibody
    • spondin 1 (f spondin) extracellular matrix protein antibody
    • Spondin 1 antibody
    • Spondin 1 extracellular matrix protein antibody
    • Spondin 1 precursor antibody
    • Spondin1 antibody
    • Vascular smooth muscle cell growth promoting factor antibody
    • VSGP antibody
    • VSGP/F spondin antibody
    see all

Images

  • E coli derived fusion protein as test antigen. Ab14271 dilution: 1:2000, Goat anti-IgY-HRP dilution: 1:1000. Colorimetric method for signal development.

    E coli derived fusion protein as test antigen. Ab14271 dilution: 1:2000, Goat anti-IgY-HRP dilution: 1:1000. Colorimetric method for signal development.

References

This product has been referenced in:
  • Jiao TT  et al. Importance of spondin 1 and cellular retinoic acid binding protein 1 in the clinical diagnosis of ovarian cancer. Int J Clin Exp Pathol 6:3036-41 (2013). IHC-P ; Human . Read more (PubMed: 24294399) »
  • Clemitson JR  et al. Genetic dissection of a blood pressure quantitative trait locus on rat chromosome 1 and gene expression analysis identifies SPON1 as a novel candidate hypertension gene. Circ Res 100:992-9 (2007). Read more (PubMed: 17332427) »
See all 2 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (Kidney)
Loading amount
20 µg
Specification
Kidney
Blocking step
Milk as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 5%

Dr. Andrew Bingham

Verified customer

Submitted Nov 14 2006

Question

This is the second order of ab14271 by the same customer. The first one worked very well. After using out , the customer ordered second. But unfortunitly there are no bands. ab14271-50 1. Order details: Batch number: lot number:119038 a.. Abcam order or Purchase order number: 95465 b.. Antibody storage conditions (temperature/reconstitution etc) -20? 2. Please describe the problem (high background, wrong band size, more bands, no band etc). No band 3. On what material are you testing the antibody in WB? Species: Mouse Cell extract or Nuclear extract: Cell Extract 3. The lysate a.. How much protein was loaded: 50?g and 100?g a.. What lysis buffer was used: M-PER Mammalian protein Extraction Reagent ( PIERCE ) a.. What protease inhibitors were used: ( sigma ) b.. What loading buffer was used: We prepared ourself c.. Did you heat the samples: temperature and time: 99? 10mins 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: Reducing Gel b.. Gel percentage : 12 % c.. Transfer conditions: NC membrane and 125mA 2 hours 5. Blocking conditions a.. Buffer: 5 % milk c.. Incubation time: 2 hours d.. Incubation temperature: Room Temperature 6. Primary Antibody a.. Specification (in which species was it raised against): Cross-reacts with Human, Mouse, Rat, and Cow At what dilution(s) have you tested this antibody: 1 : 2000 What dilution buffer was used: PBS Incubation time: 24 hours ( over-night ) Incubation temperature: 4 ? What washing steps were done: PBST 10 min 7. Secondary Antibody a.. Specification (in which species was it raised against)? Goat anti-Chicken IgY b.. At what dilution(s) have you tested this antibody: 1:10000 c.. Incubation time 1 hours d.. Wash steps: PBST 10 min e.. Do you know whether the problems you are experiencing come from the secondary? If you add too much amount of secondary antibody, it will be the high background. 8. Detection method ECL Enhance reagent ( PIERCE ) 9. Background bands Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): We don’t have any background bands. Is the blocking step sufficient? Yes, we block in 5% milk until two hours. Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) PBST 10min/time, 4 times 11. Did you apply positive and negative controls along with the samples? Please specify. We did the negative controls. And when we change the new primary antibody, we can see bands showed in image. 10. Optimization attempts How many times have you tried the Western? Four times Do you obtain the same results every time e.g. are background bands always in the same place? No bands in every time. What steps have you altered? Add the primary antibody amount.

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Answer

Thank you for taking the time to provide all the protocol details, it was very useful to understand the problem. Could you please tell me the batch number of the first order, we have several records of selling to GeneResearch ab14271 in the last few months and I would like to compare the two lots to see if they come from the same master stock. As soon as I have this information I will be able to arrange a replacement vial and try to send the lot that worked for the customer, Thank you in advance,

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Question

BATCH NUMBER 119038 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No staining SAMPLE Rat tissue. Am trying to get antibody working so have been using Kidney tissue as a positive control. PRIMARY ANTIBODY Anti SPon1 ab14271 (abcam) Diluted 1/150 in PBS Overnight incubation at 4 degrees/or RT for 1 hour Wash 3 3mins in PBS after incubation DETECTION METHOD Using a fluorescence microscope to detect FITC POSITIVE AND NEGATIVE CONTROLS USED I am using Kidney at the moment since this should detect F-spondin. This will then be my positive control except I cannot get the antibody working in the first place. ANTIBODY STORAGE CONDITIONS Aliqouted into 5ul aliquots on arrival and put into -20 freezer FIXATION OF SAMPLE Cryosectioned sample, 7um sections then let air dry before fixing. Have tried the following fixes Methanol 10 mins Methanol/Acetone 1:1 10 mins Acetone 10 mins 4% PFA in PBS 15 mins 4% PFA in PBS followed by Triton-X step 15 mins No fixation. Left primary on overnight at 4 degrees ANTIGEN RETRIEVAL None since using frozen sections PERMEABILIZATION STEP Used 0.4% Triton X mixed with block on one of the PFA fixes BLOCKING CONDITIONS Rabbit serum, 5% for 30 minutes SECONDARY ANTIBODY Anti IGY FITC ab6749 (abcam) Rabbit polyclonal to chicken iGy Diluted 1/500 in PBS Incubate 1 hour at Room temperature wash in PBS 3 x 3mins afterwards HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Fixation, I have tried various different fixes ALso, once I have left the antibody overnight at 4 degrees whilst the other time I kept it at room temperature for one hour ADDITIONAL NOTES I am using the secondary which was recommened to use on the abcam website to try and detect the spondin.

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Answer

I'm sorry to hear you are experiencing a problem with ab14271. Thank you for providing so many details about your protocol, I was very useful for me to understand your problem and I am confident that there the antibody is at fault. Unfortunately, I have found out that the IHC application on the datasheet was a mistake and it had not been tested, I am very sorry about this and can issue you a refund or credit note immediately if you could please provide your order details. Should you wish to consider leaving a negative review about this antibody please do not hesitate to do so, by clicking on the reviews tab on the online datasheet, we will offer 100 points per review and welcome all positive and negative feedback. Once again I'm very sorry about this and I look forward to hearing from you,

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Answer

Thank you for your e-mail. From the Swiss prot information the reelin domain is from aa 29-194 of spondin 1. However the immunogen sequence is located towards the carboxy end of the protein (it is located at aa 549-807 and the whole protein is 807aa long) therefore the antibody does not recognise the reelin domain. Please do not hesitate to contact us again if you need further assistance,

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Answer

Thank you for your enquiry. Please find the information regarding this protein from this website: http://au.expasy.org/cgi-bin/niceprot.pl?P35446

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Answer

Thank you for your enquiry. I will attempt to answer your enquiries point by point. 1) Where 20ug is denoted as the loading amount on a WB blot this denotes total protein and not purified target protein. 100ng in the on line protocol refers to the purified protein. 2) Please see above 3) The process of tissue extraction and homogenisation should be carried out as quickly as possible and on ice. The more time the tissue is left the more prone it is to degradation and the less feasible it will be. Transport from the animal unit should be fine (on ice) but time in transport should be kept to an absolute minimum. Protease inhibitors should also be present in the homogenisation buffer. RIPA Base Ingredients Tris-HCl: 50 mM, pH 7.4 NP-40: 1% Na-deoxycholate: 0.25% NaCl: 150 mM EDTA: 1 mM PMSF: 1 mM Aprotinin, leupeptin, pepstatin: 1 microgram/ml each RIPA Protease Inhibitors Phenylmethylsulfonyl fluoride (PMSF) (200 mM stock solution in isopropanol; store at room temperature) EDTA (calcium chelator; 100 mM stock solution in H2O, pH 7.4) Leupeptin (store frozen in aliquots, 1 mg/ml in H2O) Aprotinin (store frozen in aliquots, 1 mg/ml in H2O) Pepstatin (store frozen in aliquots, 1 mg/ml in methanol) General homogenisation protocols should be availbe from your lab, alternatively it may be possible to find these from:

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Answer

Thank you for you inquiry. Once an order has been placed for this antibody, it takes approximately 7 to 10 days for it to come into stock. Please contact us again if you have any other questions regarding our products.

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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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