Overview

  • Product name
    Anti-Bmi1 antibody [F6] - ChIP Grade
    See all Bmi1 primary antibodies
  • Description
    Mouse monoclonal [F6] to Bmi1 - ChIP Grade
  • Host species
    Mouse
  • Specificity
    Recognizes mouse Bmi-1(triplet). The clone number has been updated from (1.T.21) to (F6) both clone numbers name the same antibody clone.
  • Tested applications
    Suitable for: ICC/IF, Flow Cyt, ChIP, WB, ICC, IP, IHC-Fr, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Cat, Human, Xenopus laevis, Zebrafish
  • Immunogen

    Recombinant fragment, corresponding to amino acids 1-202 of Mouse Bmi1

  • Positive control
    • U2OS whole cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab14389 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 0.65 µg/ml. PubMed: 19001505
Flow Cyt Use a concentration of 0.65 µg/ml. PubMed: 19001505

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ChIP Use at an assay dependent concentration. PubMed: 18332116
WB Use a concentration of 0.2 - 2 µg/ml. Predicted molecular weight: 37 kDa. Detects Bmi-1 in RIPA lysates from U2OS cells. U2OS cell lysate was resolved by electrophoresis, transferred to nitrocellulose and probed with ab14389 0.2ug/ml. Proteins were visualized using a goat anti-mouse IgG labeled with HRP and a chemiluminescence detection system.
ICC Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration. PubMed: 17210912
IHC-P 1/100.

Target

  • Function
    Component of the Polycomb group (PcG) multiprotein PRC1 complex, a complex required to maintain the transcriptionally repressive state of many genes, including Hox genes, throughout development. PcG PRC1 complex acts via chromatin remodeling and modification of histones; it mediates monoubiquitination of histone H2A 'Lys-119', rendering chromatin heritably changed in its expressibility. In the PRC1 complex, it is required to stimulate the E3 ubiquitin-protein ligase activity of RNF2/RING2.
  • Sequence similarities
    Contains 1 RING-type zinc finger.
  • Post-translational
    modifications
    Monoubiquitinated (By similarity). May be polyubiquitinated; which does not lead to proteasomal degradation.
  • Cellular localization
    Nucleus. Cytoplasm.
  • Information by UniProt
  • Database links
  • Alternative names
    • B lymphoma Mo MLV insertion region (mouse) antibody
    • B lymphoma Mo MLV insertion region 1 homolog antibody
    • Bmi 1 antibody
    • BMI1 antibody
    • BMI1 polycomb ring finger oncogene antibody
    • BMI1_HUMAN antibody
    • Flvi 2/bmi 1 antibody
    • FLVI2/BMI1 antibody
    • MGC12685 antibody
    • Murine leukemia viral (bmi 1) oncogene homolog antibody
    • Oncogene BMI 1 antibody
    • PCGF 4 antibody
    • PCGF4 antibody
    • Polycomb complex protein BMI 1 antibody
    • Polycomb complex protein BMI-1 antibody
    • Polycomb group protein Bmi1 antibody
    • Polycomb group ring finger 4 antibody
    • Polycomb group RING finger protein 4 antibody
    • RING finger protein 51 antibody
    • RNF 51 antibody
    • RNF51 antibody
    see all

Images

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: Bmi1 knockout HAP1 cell lysate)
    Lane 3: U2OS cell lysate (40 ug)
    Lane 4: Molt-4 cell lysate (40ug)
    Lanes 1 - 4: Merged signal (red and green). Green - ab14389 observed at 42 kDa. Red - loading control, ab176560, observed at 52 kDa.

    ab14389 was shown to recognize Bmi1 when Bmi1 knockout samples were used, along with additional cross-reactive bands. Wild-type and Bmi1 knockout samples were subjected to SDS-PAGE. ab14389 at a concentration of 1 μg/ml and ab176560 (loading control to alpha tubulin) diluted to 1/10000 were incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • SK-N-SH cells were fixed in 4% paraformaldehyde, permeabilized in 0.55 Triton-X100 and incubated for 1 hour with ab14389 (1/200). The Bmi1 staining is shown in red. The cells were counterstained with DAPI (blue). 100x magnification.

  • Cells were lysed in RIPA buffer. Samples (25 µg) of total protein were separated by SDS-polyacrylamide gel electrophoresis gel (PAGE) in a 12% gel and transferred onto a nitrocellulose membrane. This was followed by an incubation with mouse anti-BMI1 (ab14389) and a sheep anti-mouse HRP conjugated antibody diluted 1:2000 was used as a secondary antibody. HRP conjugated anti-GAPDH antibody (ab9482) was used as a loading control.

    The mCbx7 expressing cells were infected with lentivirus-based shRNA vectors targeting BMI1 or an irrelevant control (C).

  • Formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues and normal colorectal tissues. Endogenous peroxidase was blocked by incubating the sections in a 0.3% solution of hydrogen peroxide (in PBS) for 20 min. Antigen retrieval was performed by heating the sections for 10 min at 95°C in a citrate buffer. Tissues were incubated with ab14389 overnight (16 hrs). Staining was visualized using the Dako REAL EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse (Dako). The stained TMA sections were scanned using a 20x magnification on the semi-automated Ariol system (Leica Microsystems, Wetzlar, Germany). Tumor cell areas (tumor tissues) and colon epithelium (in normal tissues) were identified for positive (indicated by yellow dots) and negative (blue dots) nuclei in tumor cores. TMA slides were scanned using a 20x magnification. Shown are positively stained tumor cores (top image) and negative tumor cores (bottom image).

  • Western blot analysis of RIPA lysates from U2OS cells labelling Bmi-1 with ab14389 at 0.2μg . An IgG (HRP) goat anti-mouse was used as the secondary antibody.

     

  • ab14389 staining Bmi1 in human head and neck squamous cell cancers (HNSCC) by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
    Sections were deparaffinized in xylene, dehydrated through graded alcohols, and placed in 0.1% hydrogen peroxide to quench any endogenous peroxidase activity. A 5 minute, 750 W microwave pretreatment in citrate buffer (pH 6.0) was repeated 4 times and followed by treatment with 10% normal rabbit serum for 30 minutes to block nonspecific antibody binding. The slides were then incubated with ab14389 at a 1/100. Primary antibody incubation was followed by three washes with PBST and incubation with fluorescently-labeled Cy2 (1/250) secondary antibody for 2 hours. Nuclei were counterstained with DAPI. Finally, sections were rinsed with PBST, dehydrated through sequential washes in 50%, 70%, 95%, and 100% ethanol and then cleared in xylene. Slides were mounted with DPX mounting media and allowed to dry overnight.

References

This product has been referenced in:
  • Sun ZJ  et al. A fusion protein composed of the DSL domain of Dll1 and RGD motif protects cryptic stem cells in irradiation injury. Biosci Rep 38:N/A (2018). Read more (PubMed: 29444821) »
  • Chow HY  et al. Group I Paks are essential for epithelial- mesenchymal transition in an Apc-driven model of colorectal cancer. Nat Commun 9:3473 (2018). Read more (PubMed: 30150766) »
See all 55 Publications for this product

Customer reviews and Q&As

1-10 of 28 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (differentiated induced pluripotent stem cells)
Permeabilization
Yes - 0.25% Triton X-100 in PBS
Specification
differentiated induced pluripotent stem cells
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 22°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jul 19 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rhesus monkey Tissue sections (Jejunum)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Ventana CC1 (High pH)
Permeabilization
No
Specification
Jejunum
Fixative
Paraformaldehyde

Dr. Li Li

Verified customer

Submitted May 31 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (human acute promylocyitc leukemia NB4 cells)
Loading amount
40 µg
Specification
human acute promylocyitc leukemia NB4 cells
Treatment
1 uM ATO treatment for indicated time
Gel Running Conditions
Reduced Denaturing (10% gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Mr. Sungsin Jo

Verified customer

Submitted Apr 02 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Brain)
Specification
Brain
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate buffer pH6
Permeabilization
No
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 7.5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Oct 02 2012

Answer

Thank you for contacting us.

I am sorry that this antibody did not perform as stated on the datasheet. As requested, I have asked our Finance department to issue a credit note for you.

Credit note ID : 22394


The credit note may be used in one of the following ways:

(1) Redeemed against the original invoice if this hasn't already been paid.
(2) Held on the account for use against a future order.
(3) A full refund can be offered where no other invoices are outstanding.

Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website.

The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

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Answer

Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note. This can then be redeemed against the invoice of a future order.



As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

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Answer

Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note for ab53673 Anti-AREB6 antibody [416A7H10]. This can then be redeemed against the invoice of a future order.

Credit ID: 123456

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

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Answer

Thank you for taking the time to complete our questionnaire. The details you have kindly provided will provide us with vital information for our monitoring of product quality

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been succesful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably recieved a bad vial.

I apologise for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation (providing the product has been purchased in the last 6 months). In order to arrange this, I would appreciate if you could confirm your order number and date of purchase?

Thank you for your continued cooperation. I look forward to hearing from you with details of how you would like to proceed.

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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 this antibody.

I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. Also, this antibody is tested and covered by our 6 month guarantee forIHC-P and IHC-Frand in Mouse, Rat, Rabbit, Cat, Human andZebrafish samples. In the event that a product is not functioning in the tested applications and species 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.

It is regrettable this second vial has not worked for you and I fully understand your concerns. I would like to investigate this case further, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you could also provide an image (from the lot that worked and the one that hasn't) which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire and hope we can resolve this case as soon as possible for you.

Order Details
Antibody code:

Lot number:

Purchase order number
or preferably Abcam order number:


General Information
Antibody storage conditions (temperature/reconstitution etc)

Aliquoted and stored the antibody at -20 degrees upon arrival

Description of the problem (high background, low signal, non-specific satining etc.)

Sample (Species/Tissue/Cell Type/Cell Line etc.)

Colorectal cancer tissue (which species?)

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)


Antigen retrieval (Enzymatic method, Heat mediated technique etc.)


Permeabilization step


Blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

1:200,1:100 and even 1:50

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method


Positive and negative controls used (please specify)


Optimization attempts (problem solving)
How many times have you tried the IHC?


Have you run a No Primary control?
Yes No

Is the current vial of secondary antibody working well with other primary antibodies?

Do you obtain the same results every time?
Yes No


What steps have you altered?


Additional Notes

This antibody has been used previously on breast tissue TMAs (same antibody, different lot) and this has worked fine at a final dilution of 1:200. Previous vial showed sufficient results in a first test round using tonsil as a positive control and testing this antibody on colorectal cancer tissue (dilution 1:200).


We would appreciate if you are also able to provide and image which would help us to assess the results

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Question

- Antibody storage conditions (temperature/reconstitution etc) -20℃ 2. Please describe the problem (high background, wrong band size, more bands, no band etc). No band. 3. On what material are you testing the antibody in WB? - Species: human - What’s cell line or tissue Tw01 - Cell extract or Nuclear extract: cell extract - Purified protein or Recombinant protein: 3 The lysate - How much protein was loaded: 40μg - What lysis buffer was used: RIPA buffer - What protease inhibitors were used: ProBlockTM Gold Mammalian Protease Inhibitor Cocktail [100X] (Catalog ID: GB-331-1) - What loading buffer was used: 6x sample buffer - Phosphatase inhibitors GenScript Cat: HC100-008 - Did you heat the samples: temperature and time: yes 95℃, 5 min 4. Electrophoresis/Gel conditions/ Transfer conditions - Reducing or non reducing gel: Reducing - Reducing agent: SDS - Gel percentage : 10% - Transfer conditions: (Type of membrane, Protein transfer verified): NC paper(0.22μm) - 5. Blocking conditions - Buffer: - Blocking agent: milk, BSA, serum, what percentage:5%, milk - Incubation time:1 hour - Incubation temperature: room temperature 6. Primary Antibody :ab14389 - Species:mouse - Reacts against: human - At what dilution(s) have you tested this antibody:1:1000(ab14389, ab53673), 1:50(ab50887) - What dilution buffer was used: ) - Incubation time: over night - Incubation temperature: 4℃ - What washing steps were done: 10mins/once, three times 7. Secondary Antibody - Species:goat - Reacts against: mouse - At what dilution(s) have you tested this antibody: 1:10000 - Incubation time: 1 hour - Wash steps: 10mins/once, three times - Fluorochrome or enzyme conjugate:enzyme conjugate - Do you know whether the problems you are experiencing come from the secondary? 8. Detection method ECl, ECl+, other detection method: ECL+ 9. Did you apply positive and negative controls along with the samples? Please specify. Yes, α-Tubulin 10. Optimization attempts - How many times have you tried the Western? twice

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

I have checked the datasheet for the ab53673 Anti-AREB6 antibody [416A7H10], and unfortunately, it looks like the antibody hasn't been tested on the endogenous protein yet. I want to offer your customer a credit note or a free of charge antibody on this occasion.

Nonetheless, having reviewed this case, I would like to offer some suggestions to help optimize the results from ab14389 Anti-Bmi1 antibody [1.T.21] - ChIP Grade and ab50887 Anti-Twist antibody [Twist2C1a] - ChIP Grade:


1. Please be aware that alpha-Tubulin is a cytoplasmatic loading control, but both targets are located rather nuclear. If you want to use a nuclear loading control, I would like to suggest to use an Anti-TATA binding protein TBP antibody as nuclear loading control.


2. According to the datasheets it looks like both antibodies need a relatively high antibody concentration, high protein load and a long exposition time. This means that the targets are probably not very abundantly expressed. In order to concentrate the protein I would suggest to perform a nuclear fraction protocol.


3. Please increase the dilution for the ab14389 up to 1:500, and expose the film 10min prior developing.


4. The same for ab50887: Please expose the film 10min prior developing .


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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1-10 of 28 Abreviews or Q&A

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