Overview

  • Product name

  • Description

    Rabbit polyclonal to CYP1B1
  • Host species

    Rabbit
  • Tested applications

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

    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Human CYP1B1 aa 1-100 conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab33584, ab33585)

  • Positive control

    • Ab32649 gave a positive signal in the following human tissue lysates: Brain; Kidney; Liver. This antibody gave a positive result in IF in the following formaldehyde fixed cell lines: HeLa.

Properties

Applications

Our Abpromise guarantee covers the use of ab32649 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 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 70 kDa (predicted molecular weight: 61 kDa).
IHC-P 1/1500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function

    Cytochromes P450 are a group of heme-thiolate monooxygenases. In liver microsomes, this enzyme is involved in an NADPH-dependent electron transport pathway. It oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics.
    Participates in the metabolism of an as-yet-unknown biologically active molecule that is a participant in eye development.
  • Tissue specificity

    Expressed in many tissues.
  • Involvement in disease

    Defects in CYP1B1 are the cause of primary congenital glaucoma type 3A (GLC3A) [MIM:231300]. GLC3A is an autosomal recessive form of primary congenital glaucoma (PCG). PCG is characterized by marked increase of intraocular pressure at birth or early childhood, large ocular globes (buphthalmos) and corneal edema. It results from developmental defects of the trabecular meshwork and anterior chamber angle of the eye that prevent adequate drainage of aqueous humor.
    Defects in CYP1B1 are a cause of primary open angle glaucoma (POAG) [MIM:137760]. POAG is a complex and genetically heterogeneous ocular disorder characterized by a specific pattern of optic nerve and visual field defects. The angle of the anterior chamber of the eye is open, and usually the intraocular pressure is increased. The disease is asymptomatic until the late stages, by which time significant and irreversible optic nerve damage has already taken place. In some cases, POAG shows digenic inheritance involving mutations in CYP1B1 and MYOC genes.
    Defects in CYP1B1 are a cause of Peters anomaly (PAN) [MIM:604229]. Peters anomaly is a congenital defect of the anterior chamber of the eye.
  • Sequence similarities

    Belongs to the cytochrome P450 family.
  • Cellular localization

    Endoplasmic reticulum membrane. Microsome membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • Aryl hydrocarbon hydroxylase antibody
    • CP1B antibody
    • CP1B1_HUMAN antibody
    • Cyp1b1 antibody
    • CYPIB1 antibody
    • Cytochrome P450 1B1 antibody
    • Cytochrome P450 family 1 subfamily B polypeptide 1 antibody
    • Cytochrome P450 subfamily I (dioxin inducible) polypeptide 1 (glaucoma 3 primary infantile) antibody
    • Flavoprotein linked monooxygenase antibody
    • GLC3A antibody
    • Microsomal monooxygenase antibody
    • P4501B1 antibody
    • Xenobiotic monooxygenase antibody
    see all

Images

  • All lanes : Anti-CYP1B1 antibody (ab32649) at 1 µg/ml

    Lane 1 : Brain (Human) Tissue Lysate - adult normal tissue
    Lane 2 : Kidney (Human) Tissue Lysate - adult normal tissue
    Lane 3 : Liver (Human) Tissue Lysate - adult normal tissue

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 61 kDa
    Additional bands at: 70 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 30 seconds
  • Image courtesy of Human Protein Atlas

    ab32649 staining CYP1B1 in human stomach. Paraffin embedded human stomach tissue was incubated with ab32649 (1/1500 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

    ab32649 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

    .
  • ICC/IF image of ab32649 stained HeLa cells. The cells were 4% formaldehyde fixed (5 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 ab32649 at 1µg/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.

References

This product has been referenced in:

  • Xie S  et al. CXCR4 promotes cisplatin-resistance of non-small cell lung cancer in a CYP1B1-dependent manner. Oncol Rep 37:921-928 (2017). Read more (PubMed: 27922681) »
  • Johansen AK  et al. The serotonin transporter promotes a pathological estrogen metabolic pathway in pulmonary hypertension via cytochrome P450 1B1. Pulm Circ 6:82-92 (2016). IHC-P . Read more (PubMed: 27162617) »
See all 7 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

Thank you for confirming the order number. Unfortunately the order has been placed in December 2012 which means the product is out of 6 month guarantee period so this purchase order is not eligible for replacements or refunds. Please click the link for more information about the guarantee; https://www.abcam.com/index.html?pageconfig=abpromise

I am however sending you this replacement product as one time exception. Please do check Abcam's guarantee policy.

The order number is 1125919. Please note the product is currently out of stock and will be available for dispatch on 17th July.

I hope the new product will work just fine; if however you experience any problem, please do not hesitate to contact us.

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Answer

I am sorry to hear that the results isn't improved.

In order to dispatch the replacement as you requested I would like to know the purchase order number. Please contact your accounts department; they will be able to give you this information.

I will look forward to hearing from you.

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Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

As we discussed please try the antibody at 1/1000 or 1-5ug/ml dilution.

Thank you very much for your cooperation. Please let me know how the antibody performs.

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Answer

As requested I have processed the refund for ab32649, your credit note number is ******. If there is anything else I can help you with, please let me know. I hope that you are able to find another antibody that will give you a cleaner signal in your western blots and so you can continue with your research.

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Question

Hi, I'm quite disappointed by your response since the details you ask for are in my original submission; but I will answer them again below. 1 - Are you seeing any bands at 70kDa, which could be your band of interest? If all the extra bands are below 70kDa, it could be that your sample has been digested due to insufficient protease inhibitors. I attached the blot to my submission and have attached it again to this email. There is a small band at 70kDa which could be the correct protein but it is not the largest/most intense band on the blot.  Protein degradation normally appears as smears and not as distinct bands of lower molecular weights; furthermore, the other antigens I have looked at in these samples are all giving clean singles bands (including CYP1A1).  We routinely include protease inhibitors in our lysis buffers.  Only this new antibody is giving multiple bands.  2 - The paper on our website that used this antibody, used it at a 1/20000 dilution, so maybe decreasing from 1/1000 to even 1/5000, should help get rid of none specific bands. They also did an overnight incubation, so if you drop the concentration down, I would also recommend incubating O/N. I used 1:1,000 because the data sheet Abcam supplied gives the following information: 0.4mg/mL original concentration, suggested concentration for western blotting 1ug/mL (datasheet attached for your reference).  Therefore a 1 in 400 dilution!!!  I don't know where the 1 in 20,000 dilution comes from and I can only work with the information Abcam give me.  I did incubate overnight as stated in my original submission. 3 - Also to try and get rid of background bands, you could try using 5% milk, which is compatible with the Odyssey system, as again the milk block has proved to be effective. We have tested both milk and Odyssey and they give comparable results so this is not the issue. As stated previously, a secondary only control shows no bands so blocking was perfect. 4- You say in your methods that you heated your samples to 70C, increasing the temperature to 100C for 10 minutes would help break up any complexes that CY1B1 may be in, if you are also seeing higher molecular weight bands. 70 degrees heating for 5 minutes is a standard protocol for CYP blotting and in fact boiling at 100 degrees causes aggregation of membrane associated proteins so this will not resolve this problem.  My CYP1A1 antibody gives a single specific band on the same lysates processed by the same method. I do not believe that any of these suggestions will resolve the problems we are having with this antibody and I would like a refund so I can source a more specific antibody. I look forward to your response on this matter.

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Answer

Thank you for your response. I am sorry that you were disappointed by my original response. Unfortunately the image of you results that you attached in your original email did not come through in our system and so I was unable to see the mass of non-specific bands that the antibody was generating. Therefore when I read that the antibody was giving multiple bands in you blot, I just wanted to clarify what type of banding pattern you were seeing. Also, below is a link to the reference that is available on our website for ab32649, which is where I found the 1/20000 dilution: http://www.ncbi.nlm.nih.gov/pubmed/20887769?dopt=Abstract Once again, please accept my apologies in this matter and I would be very happy to process a refund or send a replacement. If you would like to receive a refund, I believe I have found your original order, but could you please just confirm that it was ordered on******

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Question

LOT NUMBER ***** ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Multiple bands SAMPLE Human urothelial cells treated with the AhR ligand ITE, Bladder Cancer Cell Lines and Pooled Donor Human liver microsomes. PRIMARY ANTIBODY ab32649 (lot# GR39390-1) Rabbit polyclonal 1 in 1,000 diluted in TBS:Odyssey Blocking Buffer (50:50) at time of use and incubated overnight at 4 degrees. DETECTION METHOD Li-Cor scan POSITIVE AND NEGATIVE CONTROLS USED Negative control - Membrane was probed first using a mouse anti-AhR antibody with IRDye 700 secondary and in this scan there were no bands present in the 800 channel used to detect the rabbit CYP1B1 antibody. Positive control - Human liver microsomes were used and gave multiple bands suggesting cross-reactivity with other CYPs. ANTIBODY STORAGE CONDITIONS Antibody has been stored at +4 degrees since delivery SAMPLE PREPARATION Lysed in 2xSDS buffer, heated at 70degrees for 5min and processed as per manufacturers instructions for the Invitrogen NuPAGE system. AMOUNT OF PROTEIN LOADED 40ug ELECTROPHORESIS/GEL CONDITIONS Reducing, 4-12% Bis-Tris run in MOPS buffer. TRANSFER AND BLOCKING CONDITIONS Transfered in 20% methanol tris-glycine buffer for 2.5 hours at 4 degrees. SECONDARY ANTIBODY LiCor Odyssey Goat-Anti-Rabbit IRDye 800 1 in 1,000 diluted in TBS:Odyssey Blocking Buffer (50:50) at time of use and incubated for 1 hour at ambient temperature. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? Once HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes

Read More
Answer

Thank you for contacting Abcam. I am sorry that you are having problems with ab32649 in western blotting. I have a few more questions/suggestions about the protocol that you are using which could help. 1 - Are you seeing any bands at 70kDa, which could be your band of interest? If all the extra bands are below 70kDa, it could be that your sample has been digested due to insufficient protease inhibitors. 2 - The paper on our website that used this antibody, used it at a 1/20000 dilution, so maybe decreasing from 1/1000 to even 1/5000, should help get rid of none specific bands. They also did an overnight incubation, so if you drop the concentration down, I would also recommend incubating O/N. 3 - Also to try and get rid of background bands, you could try using 5% milk, which is compatible with the Odyssey system, as again the milk block has proved to be effective. 4- You say in your methods that you heated your samples to 70C, increasing the temperature to 100C for 10 minutes would help break up any complexes that CY1B1 may be in, if you are also seeing higher molecular weight bands. I hope that some of these protocol tips will prove helpful and I look forward to your reply.  

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Application
Western blot
Sample
Human Tissue lysate - whole (endometrial cancer and adjacent control endometriu)
Loading amount
60 µg
Specification
endometrial cancer and adjacent control endometriu
Gel Running Conditions
Reduced Denaturing
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 Feb 08 2011

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (endometrial cancer tissue)
Specification
endometrial cancer tissue
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: sodium citrate buffer in microwave oven
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 08 2011

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