Overview

  • Product name
  • Description
    Rabbit polyclonal to PUMA
  • Host species
    Rabbit
  • Specificity
    At least 2 isoforms are known to exist; this antibody will detect both isoforms.
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide:

    PLPRGHRAPEMEPN

    , corresponding to C terminal amino acids 180-193 of Human PUMA alpha. (Peptide available as ab9644.)

  • Positive control
    • K562 or 3T3
  • General notes


    Apoptosis is related to many diseases and development. The p53 tumor-suppressor protein induces apoptosis through transcriptional activation of several genes. A novel p53 inducible pro-apoptotic gene was identified recently and designated PUMA (for p53 upregulated modulator of apoptosis) and bbc3 (for Bcl-2 binding component 3) in human and mouse (1-3). PUMA/bbc3 is one of the pro-apoptotic Bcl-2 family members including Bax and Noxa, which are also transcriptional targets of p53. The PUMA gene encodes two BH3 domain-containing proteins termed PUMA-a and PUMA-b (1). PUMA proteins bind Bcl-2, localize to the mitochondria, and induce cytochrome c release and apoptosis in response to p53. PUMA may be a direct mediator of p53-induced apoptosis.

Properties

Applications

Our Abpromise guarantee covers the use of ab9643 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 Use a concentration of 1 - 2 µg/ml. Detects a band of approximately 23 kDa.Can be blocked with Human PUMA peptide (ab9644). Use at a concentration of 1 - 2 µg/ml. Detects a band of approximately 23 kDa. Can be blocked with PUMA peptide (180/193) (ab9644). A lower band at approximately 16 kDa was detected in MOLT4 and U937 cells, which may represent the PUMA-beta form.
IHC-P Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.

Target

Images

  • Anti-PUMA antibody (ab9643) at 1/1000 dilution + HeLa whole cell lysate

    Secondary
    Alexa Fluor 680-conjugated goat anti-rabbit IgG polyclonal at 1/1 dilution

    Observed band size: 20 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 26 kDa (possible non-specific binding), 42 kDa (possible non-specific binding), 50 kDa (possible non-specific binding)


    Exposure time: 5 seconds


    Blocked with 5% milk for 1 hour.

    Incubated with the primary antibody for 18 hours.

    See Abreview

  • ab9643 staining rat gonad tissue sections by Immunohistochemistry (IHC-P - Formalin/PFA-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 1.5% milk for 45 minutes at 25°C. Protease/PBS (100mg/200ml) was used for antigen retrieval, H2O2/methanol solution (0.3%) was used to destroy endogenous peroxidase activity. Samples were incubated with the primary antibody (1/500) for 12 hours at 4°C. A Biotin-conjugated goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

    See Abreview

  • ICC/IF image of ab9643 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 (ab9643, 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:
  • Kong YH & Xu SP Salidroside prevents skin carcinogenesis induced by DMBA/TPA in a mouse model through suppression of inflammation and promotion of apoptosis. Oncol Rep 39:2513-2526 (2018). Read more (PubMed: 29693192) »
  • Banu SK  et al. Chromium VI - Induced developmental toxicity of placenta is mediated through spatiotemporal dysregulation of cell survival and apoptotic proteins. Reprod Toxicol 68:171-190 (2017). Read more (PubMed: 27443218) »
See all 69 Publications for this product

Customer reviews and Q&As

1-10 of 18 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (human hepatocytes)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Treatment
100 ng/ml for hepatocytes
Specification
human hepatocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Apr 24 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 hepatocytes)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
human hepatocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Mar 13 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Gonad)
Antigen retrieval step
Enzymatic
Permeabilization
No
Specification
Gonad
Blocking step
Milk as blocking agent for 45 minute(s) · Concentration: 1.5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Apr 14 2015

Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (hepatocytes)
Specification
hepatocytes
Treatment
100 U/ml IL-1 beta
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Dec 23 2014

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (10% SDS PAGE)
Sample
Mouse Tissue lysate - whole (Liver)
Specification
Liver
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jul 22 2014

Answer

Thank you for contacting us.


I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

The credit note ID is for your reference only and does not automatically guarantee the credit.

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.

Read More

Answer

Thank you for contacting Abcam.

You are correct that you should be seeing bands at 23 &16kDa. I am wondering if the bands that you are seeing at 56 & 72kDa is due to the protein being in a complex with other proteins.

How long do you boil your samples for? As increasing the boiling time to 10 minutes, maybe even longer will help to break up these complexes and allow the protein to run at its normal molecular weight.

Let me know if you have already tried this or if you try it and it does not solve the problem. The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot on human samples. If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

I look forward to your reply.

Read More

Answer

Thank you for contacting Abcam. 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 replacement from a new lot. 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.  

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - whole (Liver)
Loading amount
40 µg
Specification
Liver
Treatment
2Hrs post 6Gy IR
Gel Running Conditions
Reduced Denaturing (12% gel)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Sep 26 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (brain)
Loading amount
25 µg
Specification
brain
Treatment
saline 0.9%-6 hrs, ethanol 20%-6 hrs
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 27°C

Arindam Ghosh

Verified customer

Submitted Mar 08 2011

1-10 of 18 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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