Overview

  • Product name
    Anti-Cytochrome P450 1A1 antibody
    See all Cytochrome P450 1A1 primary antibodies
  • Description
    Rabbit polyclonal to Cytochrome P450 1A1
  • Host species
    Rabbit
  • Specificity
    Detects Cytochrome P450 1A1 from Human cells. This antibody detects, to a lesser extent, other P450 isoforms.
  • Tested applications
    Suitable for: IHC-P, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Other Immunogen Type (His-tag) corresponding to Human Cytochrome P450 1A1. Purified, His-tagged, full length human P450 1A1 fusion protein.

  • Positive control
    • MCF-7 cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab3568 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
IHC-P 1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF 1/1000.
WB 1/500.

By Western blot, this antibody detects an ~54 kDa protein representing P450 1A1 from MCF-7 cell extract.

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.
  • Tissue specificity
    Lung, lymphocytes and placenta.
  • Sequence similarities
    Belongs to the cytochrome P450 family.
  • Cellular localization
    Endoplasmic reticulum membrane. Microsome membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • AHH antibody
    • AHRR antibody
    • Aryl hydrocarbon hydroxylase antibody
    • CP11 antibody
    • CP1A1_HUMAN antibody
    • CYP 1 antibody
    • CYP1 antibody
    • cyp1a1 antibody
    • CYPIA1 antibody
    • Cytochrome P1 450 dioxin inducible antibody
    • Cytochrome P1-450 antibody
    • Cytochrome P450 1A1 antibody
    • Cytochrome P450 family 1 subfamily A polypeptide 1 antibody
    • Cytochrome P450 form 6 antibody
    • Cytochrome P450 subfamily I (aromatic compound inducible) polypeptide 1 antibody
    • Cytochrome P450-C antibody
    • Cytochrome P450-P1 antibody
    • Flavoprotein-linked monooxygenase antibody
    • Microsomal monooxygenase antibody
    • P1 450 antibody
    • P450 C antibody
    • P450 form 6 antibody
    • P450 P1 antibody
    • P450DX antibody
    • Xenobiotic monooxygenase antibody
    see all

Images

  • All lanes : Anti-Cytochrome P450 1A1 antibody (ab3568) at 1/500 dilution

    Lane 1 : HepG2 with Skimmed milk
    Lane 2 : LNCaP with Skimmed milk
    Lane 3 : K562 with Skimmed milk
    Lane 4 : CaCo-2 with Skimmed milk
    Lane 5 : HeLa with Skimmed milk

    Lysates/proteins at 30 µg per lane.

    Blocking peptides at 5 % per lane.

    Secondary
    All lanes : Goat anti-rabbit (H+L), HRP conjugated at 1/5000 dilution
  • ab3568 staining Cytochrom P450 1A1 in HepG2 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/250 in 0.1% BSA) for 3 hours at room temperature (panel a). An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG (H+L) polyclonal was used as the secondary antibody (1/2000). Nuclei were stained blue with DAPI (panel b), F-actin stained with Rhodamine Phalloidin (panel c) and merged image showing cyctoplasmic localization (panel d)

  • HepG2 cells were incubated at 37°C for 24h with vehicle control (0 µM) and different concentrations of itraconazole (ab120816). Increased expression of cytochrome P450 1A1 (ab3568) in HepG2 cells correlates with an increase in nifuroxazide concentration, as described in literature.

    Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab3568 at 1/500 dilution and ab8227 at 1 µg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

  • IHC image of ab3568 staining in Human breast carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab3568, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ICC/IF image of ab3568 stained MCF7 cells. The cells were 100% methanol 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 (ab3568, 1/1000 dilution) 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.

  • ab3568 at a dilution of 1/500 staining MCF-7 cell extract by Western blot.

  • HepG2 cells were incubated at 37°C for 24h with vehicle control (0 µM) and 100 µM of fluconazole (ab141065). Increased expression of cytochrome P450 1A1 (ab3568) correlates with an increase in fluconazole concentration, as described in literature.

    Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab3568 at 1/500 dilution and ab8227 at 1 µg /ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

References

This product has been referenced in:
  • Alarcan J  et al. Metabolism of the Marine Phycotoxin PTX-2 and Its Effects on Hepatic Xenobiotic Metabolism: Activation of Nuclear Receptors and Modulation of the Phase I Cytochrome P450. Toxins (Basel) 9:N/A (2017). WB ; Human . Read more (PubMed: 28678150) »
  • Mitsui Y  et al. Functional role and tobacco smoking effects on methylation of CYP1A1 gene in prostate cancer. Oncotarget 7:49107-49121 (2016). Read more (PubMed: 27203547) »
See all 7 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Pig Tissue sections (Liver)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citratbuffer ph 6
Permeabilization
No
Specification
Liver
Blocking step
commercial blocking solution (according to the manufacturer's instructions zytomed) as blocking agent for 5 minute(s) · Concentration: 3% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted May 18 2018

Answer

Thank you for contacting us. The black dots on a blot usually indicate that the antibody is binding to the blocking reagent and it needs to be filtered. The 5% milk should be in TBST. I know you have said that you have used your protocol sucessfully with other antibodies, but maybe filtering again would improve your results. I would recommend diluting the primary in 5% milk in TBST rather than just PBST or TBST. Please let me know if this problem is not resolved by these protocol tips.

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Answer

Thank you for the Western blot images that you have provided. Ab3568 was characterized for application in Western blotting using MCF-7 cell lysate and a band at approximately 54 kDa was detected. You are having a problem with non-specific bands even with your positive control. But with the microsomes sample and the MCF-7 sample there is a band at 54 kDa along with some non-specific bands. To try to reduce this non-specific banding, I would suggest further decreasing the primary antibody concentration as well as the incubation period (try 1/2000 and incubate for 2 hrs at RT). I would also suggest loading less protein (20-30 ug should be sufficient). You may also need to decrease the amount of secondary antibody that you are using and please make sure to run a secondary-only control (omit the primary) to ensure that the non-specific bands are not due to the secondary antibody. Please let me know if this helps. If not, I would be happy to provide you with a free of charge replacement vial or a credit note or refund.

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Question
Answer

Thank you for your enquiry. The concentration of ab3568 (Cytochrome P450 1A1) is ~3.1 mg/ml. Please let me know if I can provide further assistance.

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Question
Answer

Thank you for the details that you have provided and I'm sorry to hear that you are experiencing difficulty with ab3568. You mentioned that you saw positive staining with in MCF-7 cells and with commercial CYP1A1 microsomes; with these samples did you see a band at approximately 54 kDa? Or no signal or only non-specific bands? This antibody was originally characterized for application in Western blotting using MCF-7 cell lysate, and it is recommended as a positive control. Thank you and I look forward to hearing from you. Have a nice weekend.

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Question

I finished western blotting experiment yesterday. The expression of CYP1A1 and telomerase in lung cancer cell lines were detected. There is no signal developed on the telomerase membrane. Multi-bands were developed on CYP1A1 memebrane (Please find attached pictures). The procedures are described as follows: 1) Sample protein concentration was 20 ug. Same samples were loaded indentically onto two gels. Two gels (10% precast gel) were run together. 2) Membranes transfer was successful, visualised using ponceau red. The membranes was destained completely in TBST. They were incubated separately in blocking buffer (5% non-fat milk+TBST (Bio-Rad)) overnight at 4oC with agitation. 3) Then the membranes were treated with their specific antibodies for 2 hours at 4oC with agitation (CYP1A1 1:500 in blocking buffer; Telomerase 1:500 in TBST + 1% BSA (Sigma A4503)). 4) Membranes were rinsed in TBST, repeated 3x5 minutes. 5) CYP1A1 membrane was incubated with goat anti-rabbit IgG HRP-conjugate (Upstate) (1:5000 in blocking buffer) for 80 minutes at room temperature with agitation. Telomerase membrane was incubated with goat polyclonal to mouse IgM mu-F(ab)2 Fragment (HRP) (AB5930) (1:7500 in TBST + 1% BSA) for 80 minutes at room temperature with agitation. 6) Membranes were rinsed in TBST, repeated 3x5 minutes. Bands were developed using DAB/HRP detection. In the CYP1A1 detection, the multi bands developed because the primary antibody is polyclonal, but in the HL cell line, there are more bands than the other cell lines, are they non-specific banding? Also, it seems that HL and CD cell lines have much longer bands than the others, do you think that is normal and correct? Regarding the telomerae detection, was the concentration of first and secondary antibody too low? Why the molecular weight marker (Sigma) is not visualised on this membrane but developed well on the other one (CYP1A1)? Does it because of different dilution buffer used in two detections (milk-CYP1A1 + TBST, BSA-Telomerase +TBST)? I would be very grateful if you could give me any suggestions. I lookforward to hearing from you soon. Regards,

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Answer

Thank you for your enquiry. Well done on getting your western blot to work. I have read your comments and have examined your blot images. I would like ot take each of your antibodies one at a time: ab3568 - Cytochrome P450 1A1 antibody (ab3568). You have obtained good signal using 20ug of lysate. The blot that you have e-mailed me would have fewer bands were the exposure to DAB have been reduced. We generally advise the use of chemiluminescence (ECL+). This is a more sensitive approach with which you can perform many exposures of the film to the blot. A shortened exposure would have generated significantly fewer bands. Given that you have detected bands on the blot very easily; to improve the specificity of the antibody and decrease the number of non-specific bands may I suggest that you use the antibody diluted to >1:500 e.g. 1:800 or 1:1000. This should serve to improve the recognition of the antibody for its target. I think that the reason why you are observing differences in the "length" of the bands and the number of cross reacting bands is due to the fact that the mass of the lysates that you are using are subtly different. The "longer" bands are due to diffusion of the lysates into adjacent areas and it is no consequence that there are more cross reacting bands on account of the larger mass. May I suggest that you try using the antibody diluted >1:500 and use an ECL or ECL+ detection system (provided you have a dark room), perform different exposure lengths and normalise your lysates so that you can be confident that they are all 20ug. To quantitate the mass of protein you will need to perform a Bradford assay or similar protein quant. With regards the telomerase, please can you tell me which of the antibodies that you were using e.g.abxxxx. I cannot imagine that the reason that you do not have any bands on the second blot is due to the use of BSA. BSA often gives a better, cleaner more specific blot. Can you tell me whether this blot gave a good Ponceau S staining? Were there any differences in the two blots. The transfer is the most likely problem, but if you tell me the antibody that you used I can check the datasheet to determine whether I can offer any more tips specific to the antibody e.g. dilution etc. I look forward to hearing from you with further details. I hope this information helps, please do not hesitate to contact me should you require further assistance.

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