Overview

  • Product name
  • Description
    Rabbit polyclonal to TIMP3
  • Host species
    Rabbit
  • Specificity
    ab39184 binds to TIMP3. It recognizes the glycosylated and unglycosylated forms of TIMP3, and works against native or reduced TIMP3. Ab39184 does not cross react with the other TIMP family members (TIMP1, TIMP2, TIMP4).
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-P, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide of TIMP3 based on loop #1 of the human TIMP3.

    Read Abcam's proprietary immunogen policy (Peptide available as ab41152.)

  • Positive control
    • Human and mouse TIMP3 recombinant protein.

Properties

Applications

Our Abpromise guarantee covers the use of ab39184 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
ICC/IF Use a concentration of 1 - 5 µg/ml.
WB 1/1000 - 1/5000. Detects a band of approximately 24, 30 kDa (predicted molecular weight: 24 kDa). Dilution optimised using Chromogenic detection.
IHC-P Use at an assay dependent concentration. PubMed: 20448060

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

IHC-Fr 1/200.

Target

  • Function
    Complexes with metalloproteinases (such as collagenases) and irreversibly inactivates them by binding to their catalytic zinc cofactor. May form part of a tissue-specific acute response to remodeling stimuli. Known to act on MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, MMP-14 and MMP-15.
  • Involvement in disease
    Defects in TIMP3 are the cause of Sorsby fundus dystrophy (SFD) [MIM:136900]. SFD is a rare autosomal dominant macular disorder with an age of onset in the fourth decade. It is characterized by loss of central vision from subretinal neovascularization and atrophy of the ocular tissues. Generally, macular disciform degeneration develops in the patients eye within 6 months to 6 years.
  • Sequence similarities
    Belongs to the protease inhibitor I35 (TIMP) family.
    Contains 1 NTR domain.
  • Cellular localization
    Secreted > extracellular space > extracellular matrix.
  • Information by UniProt
  • Database links
  • Alternative names
    • HSMRK222 antibody
    • K222 antibody
    • K222TA2 antibody
    • Metalloproteinase inhibitor 3 antibody
    • MIG 5 protein antibody
    • MIG5 protein antibody
    • Protein MIG 5 antibody
    • Protein MIG-5 antibody
    • SFD antibody
    • Sorsby fundus dystrophy pseudoinflammatory antibody
    • TIMP 3 antibody
    • TIMP metallopeptidase inhibitor 3 antibody
    • TIMP-3 antibody
    • TIMP3 antibody
    • TIMP3_HUMAN antibody
    • Tissue Inhibitor of Metalloproteinase 3 antibody
    • Tissue inhibitor of metalloproteinases 3 antibody
    • Tissue inhibitor of metalloproteinases3 antibody
    see all

Images

  • All lanes : Anti-TIMP3 antibody (ab39184)

    Lane 1 : Human TIMP3
    Lane 2 : Mouse TIMP3
    Lane 3 : crude control (preparation of extracellular matrix from BHK cells producing recombinant human TIMP-3)

    Predicted band size: 24 kDa
    Observed band size: 21 kDa
    why is the actual band size different from the predicted?

  • ICC/IF image of ab39184 stained HeLa 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 (ab39184, 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.

  • ab39184 staining TIMP3 in mouse fallopian tube tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde, permeabilized with PBST (PBS / 0.1% v/v Triton X-100) and blocked with 3% donkey serum / 1% BSA in PBST for 1 hour at 25°C. Samples were incubated with primary antibody (1/200 in blocking buffer) for 48 hours at 25°C. An Alexa Fluor® 488-conjugated donkey anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.

    Positive TIMP3 staining is seen in the extracellular space. The image shows DAPI counterstain, red field (autofluorescence), green field antibody staining and overlay.

    See Abreview

References

This product has been referenced in:
  • Yun J  et al. IL-32 gamma reduces lung tumor development through upregulation of TIMP-3 overexpression and hypomethylation. Cell Death Dis 9:306 (2018). Read more (PubMed: 29467412) »
  • Han M  et al. Hispidulin inhibits hepatocellular carcinoma growth and metastasis through AMPK and ERK signaling mediated activation of PPAR?. Biomed Pharmacother 103:272-283 (2018). WB ; Human . Read more (PubMed: 29656183) »
See all 29 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Uterus/ Ovary/ Fallopian tube)
Permeabilization
Yes - PBST (PBS / 0.1% v/v Triton X-100 (Sigma))
Specification
Uterus/ Ovary/ Fallopian tube
Blocking step
3% donkey serum / 1% BSA (Life Technologies) in PBST as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Fixative
Paraformaldehyde

Dr. Julia Watson

Verified customer

Submitted Jun 03 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Cell lysate - whole cell (rat vascular smooth muscle cells)
Loading amount
100000 cells
Specification
rat vascular smooth muscle cells
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Feb 05 2013

Question
Answer

Thank you for contacting us.
I have checked, but unfortunately, for these antibodies we do not have more immunogen information than what is on the datasheet. The source where we receive these 3 antibodies from, considers any immunogen information proprietary and therefore we do not have this information ourselves.
I'm very sorry about this. I know this will be very disappointing for you, but I wish I could be of more help.
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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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 this product 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 ab39184.

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.

Read More

Answer

Thank you for contacting Abcam. I can confirm there is 250mM sodium chloride in the storage buffer for ab39184. Please let me know if there is anything else I can help you with.

Read More

Question

LOT NUMBER GR41994-1 ORDER NUMBER 39180 DESCRIPTION OF THE PROBLEM Multiple bands SAMPLE lanes 1,2, 3 and 4: human lung cancer cell line (Calu6)whole cell lysate, lanes 5 and 6: normal mammary epithelial cells (MCF10A)whole cell lysate, PRIMARY ANTIBODY anti-TIMP1 (ab61224) diluted 1/200 in PBS-Tween 0,1%, incubation ON at 4 celcius washed 4 times 5 min with PBS-Tw 0,1% DETECTION METHOD Enhanced Luminol (Perkin Elmer) film X-OMAT exposed 2 and 30 min POSITIVE AND NEGATIVE CONTROLS USED We consider MCF10A cells as positive control ANTIBODY STORAGE CONDITIONS aliquot the same day it arrived at the lab and stored at -20 SAMPLE PREPARATION lanes 1,2, 3 and 4 lysing buffer: HEPES 25mM pH7,5, NaCl 150mM, MgCl2 10mM, EDTA 1mM, Trition 0,1%, Glycerol 10%, aprotinin 10microM and sodium vanadate 1mM lanes 5 and 6 lysing buffer:sodium phosphate 20mM, NaCl 150mM, Triton 1%, EDTA 5mM, PMSF 0,2mg/ml, Aprotinin 10microg/ml, leupeptin 10microg/ml, sodium vanadate 0,25mg/ml Sample buffer 2X: Tris 150mM pH 6,8, SDS 1,2%, Glycerol 30%, beta-mercaptoethanol 15%, bromophenol blue 0,0018% Protocole: samples were mixed with sample buffer 2X (1:1) and boiled for 10 min AMOUNT OF PROTEIN LOADED lanes 1,3 and 5 70microg, lanes 2,4 and 6 20microg ELECTROPHORESIS/GEL CONDITIONS 12% resolving gel for denaturing SDS-PAGE and 4% stacking gel TRANSFER AND BLOCKING CONDITIONS wet transfer with Tris-Glycine-SDS buffer with 20% methanol, 900mA for 90min Blocking: PBS-Tween 0,1%- milk 5%, 90min, RT SECONDARY ANTIBODY anti-Rabbit-HRP (Santa Cruz sc-2004) 1/10000 in PBS-Tw 0,1% 90min washed 4 times 5 min with PBS-Tw 0,1% HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? We changed sample loading buffer. The one before contained less beta-mercaptoethanol and hight molecular weigth appeared on film ADDITIONAL NOTES Is it possible that we detect dimers of TIMP1

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that antibodies ab16123, ab39184 and ab61224 are not providing satisfactory results. The details provided will enable us to investigate these cases and will provide us with vital information for monitoring product quality. Having reviewed these 3 cases, I would like to offer some suggestions to help optimize the results from ab16123, ab39184 and ab61224 : To reduce the background, I recommend to load a maximum of 20µg of samples on the gel. I would suggest to use each primary antibody at a dilution 1/1000 and to dilute each one in the blocking solution. The dilution factor can then be optimized around this value. I would also recommend to try an incubation at room temperature for 2 or 3 hours. Some antibodies bind stonger and more specifically at room temperature. In order to determine the origin of the background I would recommend to run a no primary control in order to see if the background is due to some non-specific binding by the secondary antibody. Last but not least, when testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : www.abcam.com/gapdh-antibody-hrp-loading-control-ab9385.html#GAPDH-Primary-antibodies-ab9385-3.jpg . 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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Question

LOT NUMBER Gr 8727-1 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM I'm obtaining bands above 30 kDa for ab39184 and ab39185 (TIMP antibodies). In the description of the antibodies ab39184 and ab39185 is: Anti-TIMP3 antibody - Carboxyterminal end (ab39185) WB: 1/1000 - 1/5000. Used under non reducing conditions. Detects a band of approximately 21 kDa (predicted molecular weight: 24 kDa). Anti-TIMP3 antibody - Loop 1 (ab39184) WB: 1/1000 - 1/5000. Dilution optimised using Chromogenic detection. Detects a band of approximately 24 , 30 kDa (predicted molecular weight: 24 kDa). My bands are above 30 KDa, what should I do? SAMPLE Type of sample: cell lysates of mouse PRIMARY ANTIBODY Primary antibody (If more than one was used, describe in “additional notes”): only TIMP3 (ab 39184) Concentration or dilution: 1:200 Diluent buffer: TTBS 1x (TBS 10x and distilled water) Incubation time: overnight Incubation temperature: 8ºC DETECTION METHOD ECL ANTIBODY STORAGE CONDITIONS as recommended on the datasheet. SAMPLE PREPARATION Lysis buffer: RIPA Boiling for ≥5 min? yes AMOUNT OF PROTEIN LOADED Protein loaded ug/lane or cells/lane: 30 ug/ protein ELECTROPHORESIS/GEL CONDITIONS Non reducing gel. Percentage of gel: gradient 5-20% TRANSFER AND BLOCKING CONDITIONS Type of membrane: nitrocellulose membrane Protein transfer verified: yes Blocking agent and concentration: milk 5% Blocking time: 1 hour Blocking temperature: room temperature SECONDARY ANTIBODY Secondary antibody: anti-rabbit IgG HRP conjugated ( Amersham NA934) Species: Reacts against: rabbit Concentration or dilution: 1:5000 Diluent buffer: TTBS 1x Incubation time: 1h Incubation temperature: room temperature Fluorochrome or enzyme conjugate: HRP Washing after primary and secondary antibodies: Buffer: TTBS Number of washes: primary, 3x (5 min.) secondary, 3x (15 min.) DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Concentration of antibody

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Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from these antibodies. The details you have kindly provided will enable us to investigate this case for you and also gives us vital information for our monitoring of product quality. 1) When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. For unknown reasons some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : Click here for the western blot image using ab9385 (or use the following: www.abcam.com/ab9385).   2) I am unsure if the bands you are observing are around 80 kDa, as the resolution of the higher molecular weight marker bands is suboptimal. In case no bands have run out of the gel and you count from the bottom, the TIMP3 bands could also be around 60 kDa. If that is the case they may represent multimers of the protein (32 kDa dimers were reported according to the literature) or complexes with metalloproteinases. Therefore, I would recommend boiling the samples for more than 5 minutes (e.g. 10min) which may help to reduce disulfide bridges and break up complex structures. We are happy to offer this technical support. In the event that the product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund. I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.  

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Answer

Thank you for contacting us. We have indeed in our catalog 5 unconjugated anti-interferon alpha 1 antibodies tested and guaranteed to work in Western Blot and Human samples. Please note that, unfortunately, import restrictions make the goat polyclonal antibody ab86316 unavailable to Canadian customers. The remaining 4 products are very similar. However, I would recommend : - the anti-Interferon alpha antibody clone [AE3] reference ab10077 (www.abcam.com/ab10077) or - the anti-Interferon alpha antibody clone [C10F5] reference ab8317 (www.abcam.com/ab8317). These two mouse monoclonal antibodies, protein A purified, are currently in stock, and their unit size is 250µg which makes them cheaper than the other anti-IFNA1. About the other targets, I agree with you, I would recommend - ab39184 for the detection of TIMP3, - ab61224 for TIMP1, - ab95451 (Mouse monoclonal) for Maspin. ab22354 (Rabbit polyclonal) is also a good choice, - ab16123 for CDKN2A/p16INK4a. ab54210 is also well characterised with 2 publications and 3 Abreviews (customer feedbacks). 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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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Recombinant protein (N-terminal fragment of TIMP3)
Loading amount
1 µg
Specification
N-terminal fragment of TIMP3
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Jul 04 2007

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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