Overview

  • Product name
  • Description
    Goat polyclonal to CBR1
  • Host species
    Goat
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide: HGQFVSEKRVEQW, corresponding to C terminal amino acids 265-277 of Human CBR1.

  • Positive control
    • Human Liver lysate.
  • General notes
    Principal Names - CBR1; CBR; carbonyl reductase 1; carbonyl reductase (NADPH); carbonyl reductase (NADPH) 1 Official Gene Symbol - CBR1 GenBank Accession Number – NP_001748 LocusLink ID - 873 (human) Gene Ontology terms - carbonyl reductase (NADPH) activity; metabolism; cytosol; oxidoreductase activity.

Properties

Applications

Our Abpromise guarantee covers the use of ab4148 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 5 µg/ml.
WB Use a concentration of 0.1 - 0.3 µg/ml. Predicted molecular weight: 30 kDa.Can be blocked with Human CBR1 peptide (ab22991).
IHC-P Use a concentration of 1 - 2 µg/ml. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.

Target

  • Function
    NADPH-dependent reductase with broad substrate specificity. Catalyzes the reduction of a wide variety of carbonyl compounds including quinones, prostaglandins, menadione, plus various xenobiotics. Catalyzes the reduction of the antitumor anthracyclines doxorubicin and daunorubicin to the cardiotoxic compounds doxorubicinol and daunorubicinol. Can convert prostaglandin E2 to prostaglandin F2-alpha. Can bind glutathione, which explains its higher affinity for glutathione-conjugated substrates. Catalyzes the reduction of S-nitrosoglutathione.
  • Sequence similarities
    Belongs to the short-chain dehydrogenases/reductases (SDR) family.
  • Cellular localization
    Cytoplasm.
  • Information by UniProt
  • Database links
  • Alternative names
    • 15 hydroxyprostaglandin dehydrogenase [NADP+] antibody
    • 15-hydroxyprostaglandin dehydrogenase [NADP+] antibody
    • Carbonyl reductase [NADPH] 1 antibody
    • Carbonyl Reductase 1 antibody
    • CBR 1 antibody
    • CBR1 antibody
    • CBR1_HUMAN antibody
    • CRN antibody
    • NADPH dependent carbonyl reductase 1 antibody
    • NADPH-dependent carbonyl reductase 1 antibody
    • Prostaglandin 9 ketoreductase antibody
    • Prostaglandin 9-ketoreductase antibody
    • Prostaglandin E(2) 9 reductase antibody
    • Prostaglandin-E(2) 9-reductase antibody
    • SDR21C1 antibody
    see all

Images

  • Anti-CBR1 antibody (ab4148) at 0.1 µg/ml + Human Kidney lysate at 35 µg

    Predicted band size: 30 kDa



    Detected by chemiluminescence.

  • ab4148 staining (0.2µg/ml) of Human Liver lysate (RIPA buffer, 30µg total protein per lane).  Primary incubated for 1 hour.  Detected by western blot using chemiluminescence. ab4148 staining (0.2µg/ml) of Human Liver lysate (RIPA buffer, 30µg total protein per lane). Primary incubated for 1 hour. Detected by western blot using chemiluminescence.
  • Ab4148 (3ug/ml) staining human CBR1 in human kidney by immunohistochemistry using paraffin embedded tissue.

    Microwaved antigen retrieval with citrate buffer pH 6, HRP-staining.

  • ICC/IF image of ab4148 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal donkey 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 (ab4148, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 donkey anti-goat 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.

  • Lanes 1 & 10 : Protein Marker
    Lanes 2-9 : Anti-CBR1 antibody (ab4148) at 1/1000 dilution (in PBS + (0.05%) Tween at 25°C )

    Lanes 1 & 10 : Protein Marker
    Lanes 2-5 : Whole cell lysate of human lung cancer cell line A549 at 25 µg
    Lane 6 : 1X SDS Loading Buffer
    Lane 7 : recombinant CBR1 at 0.03 µg
    Lane 8 : recombinant CBR1 at 0.07 µg
    Lane 9 : recombinant CBR1 at 0.1 µg

    Secondary
    Lanes 2-9 : An HRP-conjugated anti-goat IgG at 1/10000 dilution

    Developed using the ECL technique.

    Predicted band size: 30 kDa
    Observed band size: 30 kDa


    Exposure time: 1 minute


    Blocking Step: 5% Milk for 1 hour at 25°C

References

This product has been referenced in:
  • Zhou S  et al. RACK1 promotes hepatocellular carcinoma cell survival via CBR1 by suppressing TNF-a-induced ROS generation. Oncol Lett 12:5303-5308 (2016). Read more (PubMed: 28105239) »
  • Phillips RJ  et al. Prostaglandin pathway gene expression in human placenta, amnion and choriodecidua is differentially affected by preterm and term labour and by uterine inflammation. BMC Pregnancy Childbirth 14:241 (2014). IHC . Read more (PubMed: 25048443) »
See all 14 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Lung cancer cell line (A549))
Loading amount
25 µg
Specification
Lung cancer cell line (A549)
Gel Running Conditions
Reduced Denaturing (12% Gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 10 2011

Answer

Thank you for your enquiry. We do not seem to have an exact match where the lysate has been extracted in RIPA buffer. My understanding is that the protein is cytoplasmic, therefore a lysate extracted in a gentler buffer should be ok. I would recommend looking at the following product: ab29889 https://www.abcam.com/index.html?datasheet=29889 I hope this information helps. Please do not hesitate to contact us if you need any more advice or information.

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Question

Our customer has the problem with the antibody ab4148 in WB experiment, please check the protocol and figures.THANK YOU! 1. Order details: a.. Batch number: 321914 b.. Abcam order or Purchase order number: ab4148 c.. Antibody storage conditions (temperature/reconstitution etc) 2. Please describe the problem (high background, wrong band size, more bands, no band etc). High background and wrong band size 3. On what material are you testing the antibody in WB? · Species: human · Cell extract or Nuclear extract: all · Purified protein or Recombinant protein: purified protein 3. The lysate a.. How much protein was loaded:40ug a.. What lysis buffer was used: RIPA buffer b.. What protease inhibitors were used: leupeptin aprotinin PMSF c.. What loading buffer was used: 5xsample buffer d.. Did you heat the samples: temperature and time:95℃ 10min 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: b.. Gel percentage : 10% c.. Transfer conditions: 90v 60min 5. Blocking conditions a.. Buffer: TTBS or PBS-T (T: 10% Tween 20) b.. Blocking agent: milk, BSA, serum, what percentage:5%BSA or 5%milk c.. Incubation time:overnight 4℃ d.. Incubation temperature: 4℃ 6. Primary Antibody a.. Specification (in which species was it raised against):human · At what dilution(s) have you tested this antibody: 1:1000 1:500(as show) 1:200 1:100 · What dilution buffer was used:5%bsa or 5% milk · Incubation time:4℃ overnight · Incubation temperature: 4℃ · What washing steps were done:1Xttbp or 1xpbs (5min x 3 times) 7. Secondary Antibody a.. Specification (in which species was it raised against)?Rabbit anti-Goat HRP b.. At what dilution(s) have you tested this antibody: 1:3000~1:5000 。 This second antibody has been tried for many times a.. Incubation time 1-2hr in room temp b.. Wash steps: 15min x 3 times c.. Do you know whether the problems you are experiencing come from the secondary? impossible 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): NO · 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? 30kb · Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 33kd 40kd 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?3 times or more · Do you obtain the same results every time e.g. are background bands always in the same place? almost · What steps have you altered? As above

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Answer

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab4148 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. If the suggestions do not prove to be helpful, would you please be so kind to confirm the following items in order to help me better understand the cause of the problem. In most cases, the cause of high background is because the primary and/or secondary antibody concentration is too high and causes non-specific binding. The dilution that we have on the datasheet is a recommended starting dilution and customers are encouraged to determine the optimal concentration/dilution. You can try decreasing the primary (1:2000 ~ 1:5000) and secondary antibody concentration. Another possible cause is that the amount of protein is too much. We normally recommend using 20-40 micrograms per well but please try 20ug if you have not already done so. Increasing the washing frequency and duration would also help eliminate high background. Can you confirm that you have used Tween in your washing solution and have washed the primary antibody at least 15min x 3 times? Please try this if you have not already done so. Can you confirm you have reduced and denatured the sample in buffer containing SDS and mercaptoethanol? This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with the answers to the above. Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.

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Question

Our customer has the problem with the antibody ab4148 in WB experiment, please check the protocol and figures.THANK YOU! 1. Order details: a.. Batch number: 321914 b.. Abcam order or Purchase order number: ab4148 c.. Antibody storage conditions (temperature/reconstitution etc) 2. Please describe the problem (high background, wrong band size, more bands, no band etc). High background and wrong band size 3. On what material are you testing the antibody in WB? · Species: human · Cell extract or Nuclear extract:all · Purified protein or Recombinant protein:purified protein 3. The lysate a.. How much protein was loaded:80ug a.. What lysis buffer was used: RIPA buffer b.. What protease inhibitors were used: luepeptein aprotenin c.. What loading buffer was used: 5xsample buffer d.. Did you heat the samples: temperature and time:95℃ 5min 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: b.. Gel percentage : 10% c.. Transfer conditions: 90v 60min 5. Blocking conditions a.. Buffer: TTBS or PBS b.. Blocking agent: milk, BSA, serum, what percentage:5%BSA or 5%milk c.. Incubation time:overnight 4℃ d.. Incubation temperature: 4℃ 6. Primary Antibody a.. Specification (in which species was it raised against):human · At what dilution(s) have you tested this antibody: 1:1000 1:500(as show) 1:200 1:100 · What dilution buffer was used:5%bsa or 5% milk · Incubation time:4℃ overnight · Incubation temperature: 4℃ · What washing steps were done:1Xttbp or 1xpbs 3 times 5min 7. Secondary Antibody a.. Specification (in which species was it raised against)?Rabbit anti-Goat HRP b.. At what dilution(s) have you tested this antibody: many times c.. Incubation time 1-2hr in roomtemp d.. Wash steps: 3 times 5min e.. Do you know whether the problems you are experiencing come from the secondary? impossible 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): NO · 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? 30kb · Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 31kd 24kd 40kd 55kd 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 or more · Do you obtain the same results every time e.g. are background bands always in the same place? almost · What steps have you altered?

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Answer

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab4148 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. In most cases, the cause of multiple bands is because the amount of protein is too much. We normally recommend using 20-40 micrograms per well. Loading more than this may overload the gel, which will result in many non-specific bands. Please try this amount if you have not already done so. Another possible cause is that the primary and/or secondary antibody concentration is too high and causes non-specific binding. The dilution that we have on the datasheet is a recommended starting dilution and customers are encouraged to determine the optimal concentration/dilution. You can try decreasing the primary (1:2000 ~ 1:5000) and secondary antibody concentration or run a no-primary control (without the primary antibody). Running a no-primary control will be able to eliminate the possibility that your secondary antibody is binding non-specifically. Regarding the bands that you obtained, the bands located above the expected molecular weight band could be multimers. Please try boiling your protein in SDS-PAGE for 10 minutes rather than 5 minutes to ensure proper disruption of multimers. This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. Meanwhile, bands located below the expected molecular weight may indicate that your target protein has been digested. Please make sure that you incorporated sufficient protease inhibitors and have kept your samples on ice the whole time. Increasing the washing frequency and duration would also help eliminate high background. Can you confirm that you have used Tween in your washing solution and have washed at least 15min x 3 times? I hope the above recommendations may already help you. If you still experience problems please do not hesitate to contact me with the answers to the above questions. Also, please advice on how you would to proceed with this enquiry so that I can immediately arrange for a replacement or refund to you.

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Answer

Yes I would recommend your using ab6740 (Rabbit polyclonal to Goat IgG H&L (biotin)) to detect ab4148. This secondary antibody works very well in IHC - please see the two customer reviews accessible via the online datasheet. As you have seen, ab4148 has only been tested in WB and we do not yet know if it will work in IHC. Should you go ahead and test this antibody, please let us know how you get on.

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