Overview

  • Product name
  • Description
    Rabbit polyclonal to BNIP3
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ELISA, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    KLH conjugated synthetic peptide, selected from within aa 1-100 (BH3 domain) of human NIP3.

  • Positive control
    • Mouse brain tissue lysate and Ramos cell lysate, breast carcinoma tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab38621 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
WB 1/100 - 1/500. Detects a band of approximately 22 kDa (predicted molecular weight: 22 kDa).
ELISA 1/1000.
ICC/IF Use a concentration of 1 - 5 µg/ml.
IHC-P 1/50 - 1/100. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.

Target

  • Function
    Apoptosis-inducing protein that, which can overcome BCL2 suppression. May play a role in repartitioning calcium between the two major intracellular calcium stores in association with BCL2.
  • Sequence similarities
    Belongs to the NIP3 family.
  • Cellular localization
    Mitochondrion. Mitochondrion membrane. Coexpression with the EIB 19-kDa protein results in a shift in NIP3 localization pattern to the nuclear envelope. Colocalizes with ACAA2 in the mitochondria.
  • Information by UniProt
  • Database links
  • Alternative names
    • BCL2 Adenovirus E1B 19kDa Interacting Protein 3 antibody
    • BCL2/adenovirus E1B 19 kDa protein interacting protein 3 antibody
    • BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 antibody
    • BNIP 3 antibody
    • BNIP3 antibody
    • BNIP3_HUMAN antibody
    • NIP 3 antibody
    • NIP3 antibody
    see all

Images

  • All lanes : Anti-BNIP3 antibody (ab38621) at 1/100 dilution

    Lane 1 : Ramos cell lysate
    Lane 2 : Mouse brain tissue lysate

    Predicted band size: 22 kDa
    Observed band size: 22 kDa
    Additional bands at: 15 kDa, 60 kDa, 65 kDa. We are unsure as to the identity of these extra bands.

  • This image shows human breast carcinoma stained with ab38621 at 1/50 dilution.
  • This image shows human colon carcinoma stained with ab38621 at 5ug/200ul dilution.
  • ICC/IF image of ab38621 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab38621, 1µg/ml) 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.

References

This product has been referenced in:
  • Duval N  et al. The Bcl-2 Homology-3 Domain (BH3)-Only Proteins, Bid, DP5/Hrk, and BNip3L, Are Upregulated in Reactive Astrocytes of End-Stage Mutant SOD1 Mouse Spinal Cord. Front Cell Neurosci 12:15 (2018). WB ; Mouse . Read more (PubMed: 29440992) »
  • Zhao L  et al. Long non-coding RNA HULC promotes UVB-induced injury by up-regulation of BNIP3 in keratinocytes. Biomed Pharmacother 104:672-678 (2018). WB ; Human . Read more (PubMed: 29803927) »
See all 13 Publications for this product

Customer reviews and Q&As

Question

Dear technical support team:
This customer has purchased ab38621 (Anti-BNIP3 antibody) and has conducted the wb with mouse sample. The results show no bands, therefore, she wants to ask for your help to modify her experiment step, could you please offer any suggestion to help her?
I also attached her data in this letter and her experiment step as follow:
1. Order details:
Batch number: XXXXX
Po: XXXXXX
Abcam product code: ab38621
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: Mouse
· What’s cell line or tissue:liver
· Cell extract or Nuclear extract: cell extract
· Purified protein or Recombinant protein: Purified protein
3. The lysate
How much protein was loaded: 88 ug
What lysis buffer was used: EBC lysis buffer
What protease inhibitors were used: protease inhibitor (sigma)
What loading buffer was used: 4X loading dye
Phosphatase inhibitors :no
Did you heat the samples: temperature and time:100℃ 10 min
4. Electrophoresis/Gel conditions/ Transfer conditions
Reducing or non reducing gel:
Reducing agent:
Gel percentage : 13%
Transfer conditions: (Type of membrane, Protein transfer verified): PVDF 100V 90 min
5. Blocking conditions
Buffer:TBST
Blocking agent: milk, BSA, serum, what percentage: 5% milk
Incubation time:1 hr ℃
Incubation temperature: RT
6. Primary Antibody
Species: Rabbit
Reacts against: human and mouse
· At what dilution(s) have you tested this antibody:1:200
· What dilution buffer was used: 5% milk in TBST
· Incubation time: overnight
· Incubation temperature: 4℃
· What washing steps were done: four time , 10 min/each
7. Secondary Antibody
Species:Goat
Reacts against:Rabbit
At what dilution(s) have you tested this antibody: 1:5000
Incubation time: 1 hr RT
Wash steps: 15 min once, 10 min once, 5 min five times
Fluorochrome or enzyme conjugate:HRP
Do you know whether the problems you are experiencing come from the secondary? NO
8. Detection method
ECl, ECl+, other detection method: ECl
9. Did you apply positive and negative controls along with the samples? Please specify. NO
10. Optimization attempts
· How many times have you tried the Western? 2 times
· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no
· Do you obtain the same results every time e.g. are background bands always in the same place? yes
· What steps have you altered? Protein loading
Could you please help this customer to solve the problem?
Thanks for your kindly help
Best regards

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from our products.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab8399.

-I would recommend the use of a positive control, as it is a good indicator of the antibody performance. Mouse brain tissue or Ramos cell lysate are recommended.

-BNIP3L is a phosphorilated protein. Milk is not recommended as a blocking agent when phospho-proteins, as the casein could break the phosphorilated positions. Instead, I would recommend using 5% BSA for 1 hour at RT, which in addition is a milder blocking agent and can help visualize the bands of interest.

- Increase the protein rate in your samples, using a mitochondrial lysate instead of a total cell lysate. RIPA buffer could be a good choice to do it.

-Increase the antibody concentration. Try 1/50 or 1/100 in an attempt to improve the results.

-For the washing process I would recommend using TBST, and perform 4 washes of 10 minutes each.

I hope this suggestions help improve the results from the antibody. Please do not hesitate to contact us for further assistance.

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