Overview

  • Product name
    Anti-Thrombospondin antibody [A65M]
    See all Thrombospondin primary antibodies
  • Description
    Mouse monoclonal [A65M] to Thrombospondin
  • Host species
    Mouse
  • Specificity
    ab23420 is highly specific for the low-calcium conformation of thrombospondin. There is no cross-reactivity with other connective tissue proteins (vitronectin, fibronectin, elastin, collagen, laminin).
  • Tested applications
    Suitable for: IP, ELISAmore details
  • Species reactivity
    Reacts with: Cow, Human
  • Immunogen

    Tissue, cells or virus corresponding to Cow Thrombospondin. Extracellular matrix material from cultured bovine corneal endothelial cells.

  • Epitope
    Epitope is only present in thrombospondin prepared in low-calcium (0.1 mmol/L) buffers.

Properties

Applications

Our Abpromise guarantee covers the use of ab23420 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
IP Use at an assay dependent concentration.
ELISA 1/200.

Target

  • Function
    Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions.
  • Sequence similarities
    Belongs to the thrombospondin family.
    Contains 3 EGF-like domains.
    Contains 1 TSP C-terminal (TSPC) domain.
    Contains 1 TSP N-terminal (TSPN) domain.
    Contains 3 TSP type-1 domains.
    Contains 8 TSP type-3 repeats.
    Contains 1 VWFC domain.
  • Information by UniProt
  • Database links
  • Alternative names
    • THBS 1 antibody
    • THBS antibody
    • Thbs1 antibody
    • Thrombospondin 1 antibody
    • Thrombospondin-1 antibody
    • TSP 1 antibody
    • TSP antibody
    • TSP1 antibody
    • TSP1_HUMAN antibody
    see all

References

This product has been referenced in:
  • He J & Bazan HE Epidermal growth factor synergism with TGF-beta1 via PI-3 kinase activity in corneal keratocyte differentiation. Invest Ophthalmol Vis Sci 49:2936-45 (2008). Read more (PubMed: 18579759) »
  • Behera MA  et al. Thrombospondin-1 and thrombospondin-2 mRNA and TSP-1 and TSP-2 protein expression in uterine fibroids and correlation to the genes COL1A1 and COL3A1 and to the collagen cross-link hydroxyproline. Reprod Sci 14:63-76 (2007). Read more (PubMed: 18089612) »
See all 2 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

Our customer had some trouble in trying these antibodies. There are conditions and results below. We would be appreciated if you could kindly check these questionnaire and give some advices for them. ab23420 1. Order details: a.. Batch number: S060511 b.. Abcam order or Purchase order number: ab23420-100 lot # 220092 c.. Antibody storage conditions (temperature/reconstitution etc) +4°C 2. Please describe the problem (high background, wrong band size, more bands, no band etc). more bands and high background 3. On what material are you testing the antibody in WB? · Species: rat normal liver, Hela and MCF7 · Cell extract or Nuclear extract: cell extract · Purified protein or Recombinant protein: No 3. The lysate a.. How much protein was loaded: 50 ug a.. What lysis buffer was used: RIPA b.. What protease inhibitors were used: cocktail (roche) c.. What loading buffer was used: sample buffer d.. Did you heat the samples: temperature and time: 95 ?, 5 min 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 8 % c.. Transfer conditions: 400 mA, 2 hr, 4 ? 5. Blocking conditions a.. Buffer: TBST b.. Blocking agent: milk, BSA, serum, what percentage: 5 % milk c.. Incubation time: 1 hr d.. Incubation temperature: 25 ? 6. Primary Antibody a.. Specification (in which species was it raised against): mouse · At what dilution(s) have you tested this antibody: 1: 100 · What dilution buffer was used: 5 % milk in TBST · Incubation time: 2 hr · Incubation temperature: 25 ? · What washing steps were done: washed with TBST 3 times 7. Secondary Antibody a.. Specification (in which species was it raised against)? goat b.. At what dilution(s) have you tested this antibody: 1:5000 c.. Incubation time 1 hr d.. Wash steps: washed with TBST 3 times e.. Do you know whether the problems you are experiencing come from the secondary? yes 8. Detection method ECl, ECl+, other detection method: ECL 9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes · Is the blocking step sufficient? yes · Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes · At what size are the bands migrating? Could they be degradation products of your target? no · Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 12% membrane(rat normal liver, Hela and MCF7), expected 128kDa 11. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts · How many times have you tried the Western? 2 times · Do you obtain the same results every time e.g. are background bands always in the same place? yes · What steps have you altered? no ab22825 PS. When our customer bought this antibody, it was not showed that this is the "Fast-track antibody" last year. 1. Order details: a.. Batch number: S060504 b.. Abcam order or Purchase order number: ab22825 lot:138126 c.. Antibody storage conditions (temperature/reconstitution etc) -20 ? 2. Please describe the problem (high background, wrong band size, more bands, no band etc). more bands and high background 3. On what material are you testing the antibody in WB? · Species: · Cell extract or Nuclear extract: cell extract · Purified protein or Recombinant protein: No 3. The lysate a.. How much protein was loaded: 50 ug a.. What lysis buffer was used: RIPA b.. What protease inhibitors were used: cocktail (roche) c.. What loading buffer was used: d.. Did you heat the samples: temperature and time: 95 ?, 5 min 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 20 % c.. Transfer conditions: 400 mA, 2 hr, 4 ? 5. Blocking conditions a.. Buffer: PBST b.. Blocking agent: milk, BSA, serum, what percentage: 5 % milk c.. Incubation time: 1 hr d.. Incubation temperature: 25 ? 6. Primary Antibody a.. Specification (in which species was it raised against): goat · At what dilution(s) have you tested this antibody: 1: 500 · What dilution buffer was used: 5 % milk in PBST · Incubation time: 2 hr · Incubation temperature: 25 ? · What washing steps were done: washed with PBST 3 times 7. Secondary Antibody a.. Specification (in which species was it raised against)? b.. At what dilution(s) have you tested this antibody: 1:5000 c.. Incubation time 1 hr d.. Wash steps: washed with PBST 3 times e.. Do you know whether the problems you are experiencing come from the secondary? yes 8. Detection method ECl, ECl+, other detection method: ECL 9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes · Is the blocking step sufficient? yes · Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes · At what size are the bands migrating? Could they be degradation products of your target? no · Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 11. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts · How many times have you tried the Western? 5 times · Do you obtain the same results every time e.g. are background bands always in the same place? yes · What steps have you altered? no

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Answer

Thank you for your patience. I have been in correspondence with the source of each of the antibodies. Unfortunately PTP4A1 antibody (ab22825) is a fast track antibody and has not been applied to a positive control as such. ab23420 has not been tested by western blotting but has been tested by ELISA against a commercially available source of Thrombospondin from Haematologic Techologies Inc., productnumber: HCTP-0200. I can tell you that this epitope is only present in thrombospondin prepared with low levels of calcium. The structure is regulated by sulfhydryl-disulfide interchange in the Carboxy-terminal Ca++ binding loops. Indeed the reducing conditions of your customers gel may indeed affect the epitope and potentially be the reason they are not getting the expected results. However, should the antibody work by WB the expected result should be one band at ~150 kDa. I am sorry I cannot be of more assistance, the lack of antibody characterization makes it difficult.

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Question

Our customer had some trouble in trying these antibodies. There are conditions and results below. We would be appreciated if you could kindly check these questionnaire and give some advices for them. ab23420 1. Order details: a.. Batch number: S060511 b.. Abcam order or Purchase order number: ab23420-100 lot # 220092 c.. Antibody storage conditions (temperature/reconstitution etc) +4°C 2. Please describe the problem (high background, wrong band size, more bands, no band etc). more bands and high background 3. On what material are you testing the antibody in WB? · Species: rat normal liver, Hela and MCF7 · Cell extract or Nuclear extract: cell extract · Purified protein or Recombinant protein: No 3. The lysate a.. How much protein was loaded: 50 ug a.. What lysis buffer was used: RIPA b.. What protease inhibitors were used: cocktail (roche) c.. What loading buffer was used: sample buffer d.. Did you heat the samples: temperature and time: 95 ?, 5 min 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 8 % c.. Transfer conditions: 400 mA, 2 hr, 4 ? 5. Blocking conditions a.. Buffer: TBST b.. Blocking agent: milk, BSA, serum, what percentage: 5 % milk c.. Incubation time: 1 hr d.. Incubation temperature: 25 ? 6. Primary Antibody a.. Specification (in which species was it raised against): mouse · At what dilution(s) have you tested this antibody: 1: 100 · What dilution buffer was used: 5 % milk in TBST · Incubation time: 2 hr · Incubation temperature: 25 ? · What washing steps were done: washed with TBST 3 times 7. Secondary Antibody a.. Specification (in which species was it raised against)? goat b.. At what dilution(s) have you tested this antibody: 1:5000 c.. Incubation time 1 hr d.. Wash steps: washed with TBST 3 times e.. Do you know whether the problems you are experiencing come from the secondary? yes 8. Detection method ECl, ECl+, other detection method: ECL 9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes · Is the blocking step sufficient? yes · Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes · At what size are the bands migrating? Could they be degradation products of your target? no · Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 12% membrane(rat normal liver, Hela and MCF7), expected 128kDa 11. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts · How many times have you tried the Western? 2 times · Do you obtain the same results every time e.g. are background bands always in the same place? yes · What steps have you altered? no ab22825 PS. When our customer bought this antibody, it was not showed that this is the "Fast-track antibody" last year. 1. Order details: a.. Batch number: S060504 b.. Abcam order or Purchase order number: ab22825 lot:138126 c.. Antibody storage conditions (temperature/reconstitution etc) -20 ? 2. Please describe the problem (high background, wrong band size, more bands, no band etc). more bands and high background 3. On what material are you testing the antibody in WB? · Species: · Cell extract or Nuclear extract: cell extract · Purified protein or Recombinant protein: No 3. The lysate a.. How much protein was loaded: 50 ug a.. What lysis buffer was used: RIPA b.. What protease inhibitors were used: cocktail (roche) c.. What loading buffer was used: d.. Did you heat the samples: temperature and time: 95 ?, 5 min 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 20 % c.. Transfer conditions: 400 mA, 2 hr, 4 ? 5. Blocking conditions a.. Buffer: PBST b.. Blocking agent: milk, BSA, serum, what percentage: 5 % milk c.. Incubation time: 1 hr d.. Incubation temperature: 25 ? 6. Primary Antibody a.. Specification (in which species was it raised against): goat · At what dilution(s) have you tested this antibody: 1: 500 · What dilution buffer was used: 5 % milk in PBST · Incubation time: 2 hr · Incubation temperature: 25 ? · What washing steps were done: washed with PBST 3 times 7. Secondary Antibody a.. Specification (in which species was it raised against)? b.. At what dilution(s) have you tested this antibody: 1:5000 c.. Incubation time 1 hr d.. Wash steps: washed with PBST 3 times e.. Do you know whether the problems you are experiencing come from the secondary? yes 8. Detection method ECl, ECl+, other detection method: ECL 9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes · Is the blocking step sufficient? yes · Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes · At what size are the bands migrating? Could they be degradation products of your target? no · Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 11. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts · How many times have you tried the Western? 5 times · Do you obtain the same results every time e.g. are background bands always in the same place? yes · What steps have you altered? no

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. PTP4A1 antibody (ab22825) is currently a fast track antibody that we cannot guarantee for the application of western blotting. We appreciate any feedback offered by way of our Abreviews scheme. I would like to direct your customer to www.abcam.com/abreviews. The order that was placed in the past 3 months was sold as a fast track placed. Checking through the records this antibody was purchased in 2005 as a standard price by Gene Research. The approach that your customer has adopted is very similar to one that I would recommend. I would however like to recommend that they try changing the blocking agent that they have been using to 3% BSA using an overnight incubation and reducing the antibody dilution to 1:50. It would also further assist me if you could send me a blot image of your customers blot so I can better understand the extraneous bands that they have been obtaining. I am sorry to hear that your customer has also been having difficulties applying Thrombospondin antibody [A65M] (ab23420). This antibody is yet to be applied using western blotting and therefore we cannot guarantee it for this purpose. However, I would recommend performing an approach akin to the one above; using BSA and reducing the dilution of the antibody. I am in touch with the originators of both antibodies in order to source information as to the most suitable positive control as I am not confident that HeLa and MCF7 cells are the best option. I will be in touch shortly. I appreciate your patience.

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Answer

Thank you for your enquiry. Ab23420 has only so far been tested for application in IHC-frozen tissue sections. It has not yet been tested with paraffin embedded sections. If you decide to go ahead and purchase this product, please let us know how you get on by submitting an Abreview and in return we will award you 50 Abcam Points, which can be redeemed on a number of rewards (a further 100 Abcam Points will be offered for an image). Please contact us again if you have any additional questions.

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