Overview

  • Product name
  • Description
    Rabbit polyclonal to Bmi1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IHC-P, WB, IHC-Fr, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Chicken, Cow
  • Immunogen

    Synthetic peptide corresponding to 15 amino acids near the center of human Bmi1.

  • Positive control
    • WB: K562 cell lysate. ICC/IF: Rat fibroblast cells; K562 cells. IHC-Fr: Rat brain tissue. IHC-P: Rat acute rheumatic heart disease (RHD) tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab38295 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 10 µg/ml.
IHC-P Use at an assay dependent concentration. PubMed: 20083111
WB Use a concentration of 0.5 - 1 µg/ml. Predicted molecular weight: 37 kDa.Can be blocked with Human Bmi1 peptide (ab39733).
IHC-Fr 1/300.
IP 1/2000.

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 : Anti-Bmi1 antibody (ab38295) at 0.5 µg/ml
    Lane 2 : Anti-Bmi1 antibody (ab38295) at 1 µg/ml
    Lane 3 : Anti-Bmi1 antibody (ab38295) at 2 µg/ml

    All lanes : K562 cell lysate

    Predicted band size: 37 kDa
    Observed band size: 33 kDa
    why is the actual band size different from the predicted?

  • ab38295 staining Bmi1 in Rat brain tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde and blocked with 5% serum for 1 hour at 25°C. Samples were incubated with primary antibody (1/300) for 10 hours at 4°C. An Alexa Fluor® 555-conjugated Donkey anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

    See Abreview

  • ab38295 staining Bmi1 in Rat fibroblast cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.01% Triton X-100 and blocked with 2.5% Goat serum for 10 minutes at 25°C. Samples were incubated with primary antibody (1/300) for 2 hour at 25°C. An Alexa Fluor® 555-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

    See Abreview

  • Immunofluorescence of BMI-1 in K562 cells using ab38295 at 20 ug/ml.

References

This product has been referenced in:
See all 22 Publications for this product

Customer reviews and Q&As

1-7 of 7 Q&A

Answer

Thank you for your response.
I have discussed this enquiry with my colleagues.
A BLAST with the immunogen in the human proteome does not predict cross-reactivity with any other proteins, just BMI-1 and partial BMI-1 sequences. It could be a cleavage product, but I have been unsuccessful in finding a publication that shows this.
According to Swiss-Prot database, the intracellular localisation of Bmi1 can be both nuclear and cytoplasmic. You may find some further information at this site:
http://www.uniprot.org/uniprot/P35226
1) "E4F1: a novel candidate factor for mediating BMI1 function in primitive hematopoietic cells."
Chagraoui J., Niessen S.L., Lessard J., Girard S., Coulombe P., Sauvageau M., Meloche S., Sauvageau G.
Genes Dev. 20:2110-2120(2006) [PubMed: 16882984] [Abstract]
Cited for: FUNCTION, INTERACTION WITH E4F1, SUBCELLULAR LOCATION.
2) "Interaction proteomics analysis of polycomb proteins defines distinct PRC1 Complexes in mammalian cells."
Vandamme J., Volkel P., Rosnoblet C., Le Faou P., Angrand P.O.
Mol. Cell. Proteomics 0:0-0(2011) [PubMed: 21282530] [Abstract]
Cited for: IDENTIFICATION IN A PRC1-LIKE COMPLEX, SUBCELLULAR LOCATION.
Please see attached the image of Bmi1 staining with FFPE rat thymus sections.
If you need any further assistance in the future, please do not hesitate to contact me.

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Question

Please find the questionnaire attached. In addition I add the picture of
a staining.
Best
IHC Questionnaire
It will take approximately 5 minutes to complete the questionnaire.
Please fill-in all fields so that we can better assist you
1) Abcam product code ab38295
2) Abcam order reference number or product batch number Lot: GR5707-6
3) Description of the problem
This Bmi1 antibody can detect typical “+4” cells in the small intestine.
However, the staining in these cells are mainly in the cytoplasm rather
than in the nucleus, where they should be.
4) Sample preparation:
Species: mouse
Type of sample: formalin fixed paraffin embedded sections, cells in culture
Sample preparation: stomach tissues were fixed in 4% formalin for 2
hours, followed by 1 hour’s wash in tap running water before being
dehydrated. Cells were fixed in 4% formalin for 10min before the
staining steps
Positive control : small intestine (+4 cells have been shown to be positive)
Negative control
5) Fixation step
Yes/No yes
If yes: Fixative agent and concentration 4% formalin
Fixation time : tissue:2h; cells: 10min
Fixation temperature : room temperature
6) Antigen retrieval method : retrieval was performed in boiling citrate
buffer (ph6) for 15min
7) Permeabilization method:
Did you do a permeabilization step (details please) or add
permeabilizing agent in any dilution buffers?
Permeabilizing agent and concentration:
10min incubation in 0.1-0.25% Triton-X-100(Sigma) at room temperature
8) Blocking agent (eg BSA, serum…): normal horse serum (ImmPRESS Reagent
anti-rabbit Ig peroxidase Kit, Cat.No: MP-7401)
Concentration: 2.5%
Blocking time: 30min-60min
Blocking temperature: room temperature
9) Endogenous peroxidases blocked? 3% H2O2, room temperature, 10min
Endogenous biotins blocked? Not necessary
10) Primary antibody (If more than one was used, describe in “additional
notes”) :
Concentration or dilution 1:2000
Diluent buffer PBS
Incubation time room temperature, 2h
11) Secondary antibody:
IHC:
Species: horse
Reacts against: rabbit
Concentration or dilution : from ImmPRESS Reagent anti-rabbit Ig
peroxidase Kit, Cat.No: MP-7401, it is ready to use
Diluent buffer
Incubation time: room temperature, 1h
Fluorochrome or enzyme conjugate
IF:
Species: goat
Reacts against: rabbit
Concentration or dilution: 1:200
Diluent buffer : PBS
Incubation time: room temperature, 1h
Fluorochrome or enzyme conjugate: Alexa-fluor 488
12) Washing after primary and secondary antibodies:
Buffer: PBS
Number of washes: 5min x 3
13) Detection method: AEC
14) How many times have you run this staining? About 10 times
Do you obtain the same results every time? The staining is always
predominantly in the cytoplasm
What steps have you altered to try and optimize the use of this antibody?
The concentrations of the first antibody and Triton-X-100
Document attachment: Attaching images of your IHC is strongly
recommended and can greatly speed up our investigation of your problem.

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Answer

Thank you for your enquiry regarding ab38295 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.
My colleague has asked me to look after her customers while she is busy with other projects. I apologize for my delayed response.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
1) Fixation: stomach tissues were fixed in 4% formalin for 2
hours, followed by 1 hour’s wash in tap running water before being
dehydrated.
- Question: Would you be so kind to confirm the purpose of washing the fixed tissue in tap water? Normally, for rehydration it is recommended to use TBS-T.
https://www.abcam.com/index.html?pageconfig=resource&rid=11384
https://www.abcam.com/index.html?pageconfig=resource&rid=13046
2) Antigen retrieval:
retrieval was performed in boiling citrate
buffer (ph6) for 15min
- Question: Has the time for AR been optimized and so what equipment was applied (microwave, steamer, vegetable cooker etc)?
Three minutes is only suggested as a starting point antigen retrieval time. Less than 3 minutes may leave the antigens under-retrieved, leading to weak staining. More than 3 minutes may leave them overretrieved, leading to non-specific background staining and also increasing the chances of sections dissociating from the slides. A control experiment is recommended beforehand, where slides of the same tissue section are retrieved for 1, 2, 3, 4 and 5 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.
https://www.abcam.com/index.html?pageconfig=resource&rid=11488#2
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

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Answer

Vielen Dank für Ihre Anfrage.
Es tut mir Leid zu hören, dass Sie Probleme mit diesem Antikörper haben. Laut unseren Unterlagen haben wir den Antikörper nicht selber in der IHC getestet, sondern haben diese Applikation aufgrund einer Publikation aufgenommen.
Ich habe unseren Fragebogen als Word-Dokument an diese E-Mail angehängt. Durch das Ausfüllen des Fragebogens erhalten wir alle nötigen Informationen über Ihre Proben und Ihr Protokoll. Sobald Sie dieses Formular an uns zurückgeschickt haben, werden wir uns Ihr Protokoll ansehen und möglichst Veränderungsvorschläge machen, die Ihre Ergebnisse verbessern werden.
Falls sich allerdings bestätigt, dass der Antikörper nicht so funktioniert, wie auf dem Datenblatt beschrieben und er innerhalb der letzten 180 Tage gekauft wurde, werden wir Ihnen gerne einen Ersatz oder eine Gutschrift schicken.
Ich freue mich, bald wieder von Ihnen zu hören.

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Answer

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 replacementcontaining 1 vialeach of ab38237,13533,38295.

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.

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Answer

Thank you very much for following this up with the customer. I am sorry that the change in the blocking buffer did not improve the results.

I would still like to recommend to the customer to reduce the samples for the Western blot either with DTT or beta Mercaptoethanol. Indeed, not all proteins need to be reduced to be visible in the Western blot and I am not sure whether it is necessary to reduce the samples to see a nice band of BMI1. As however the BMI1 is a highly complexed protein (http://www.uniprot.org/uniprot/P35226) and we have tested it under reduced conditions, I think there is the possibility that reducing the samples could solve the problem.

As I understand howeverthe frustration the customer must be experiencing, I will provide a credit note for this antibody. Please do let me know whether the customer would like to accept this.

Thank you for your cooperation. I am looking forward to hear back from you.

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Question

1) Abcam product code ab38295

2) Abcam order reference number or product batch number


3) Storage conditions:Store at +4°C.

4) Description of the problem (high background, no band, wrong band size...)

no band

5) Sample preparation:

Name of the tissue/cell line:hepatocellular carcinoma cell MHCC97-H

Sample species:human

Type of sample (whole cell lysates, fraction, recombinant protein…):whole cell lysates

Lysis buffer RIPA

Protease inhibitors: Aprotinin Leupeptin PMSF

Phosphatase inhibitors

Reducing agent

Boiling for ≥5 min? yes/no yes

Protein loaded ug/lane or cells/lane 40ug

Positive control NO

Negative control NO



6) Percentage of gel 10%

Type of membrane PVDF

Protein transfer verified

Blocking agent and concentration 5% milk in TBST

Blocking time 1 hour

Blocking temperature room temperature



7) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution at 1:1000 and 1:500

Diluent buffer in TBST

Incubation time overnight

Incubation temperature: at 4°C



8) Secondary antibody: Goat anti-Rabbit IgG H&L (HRP) secondary antibody
Species:Goat

Reacts against: Rabbit

Concentration or dilution 1:2000

Diluent buffer in TBST



Incubation time 2 hours

Incubation temperature:room temperature



Fluorochrome or enzyme conjugate: enzyme conjugate



9) Washing after primary and secondary antibodies:

Buffer in TBST

Number of washes every time for 10 minutes and 3 times



10)Detection method ECL



11) How many times have you run this staining? 5 times

Do you obtain the same results every time? yes

What steps have you altered to try and optimize the use of this antibody?

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Answer

Thank you for taking time to liaise with the customer and 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.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab38295. I would also appreciate if you can confirm some further details:

1.) Can you confirm that thebmi1 protein is present in theMHCC97-H cellline? (Is there for example a publication describing this?)

2.) I can stronly recommend to use also an other blocking buffer, as changing the blocking buffer can significantly improve the results. I can recommend to use BSA as an alternative blocking buffer. Please see image on the datasheet of ab9385 as an illustration.

3.) Can you please let me know whether the samples have been reduced? If not, I can recommend to do so, as the antibody has been tested under these conditions.

4.) Lastly, can you please confirm that the secondary antibody used is known to work?


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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Answer

Thank you for your enquiry. I performed a homology search of the immunogen of ab25791 and ab14399 and of the whole human protein with the drosophila protein and unfortunately found that it is so low that it is not detected by a BLAST search. I therefore do not think those antibodies will detect the Drosophila protein and recommend to purchase Drosophila specific antibodies. I'm sorry we currently do not have those in our catalogue and wish you all the best in your research,

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