Overview

  • Product name
  • Description
    Rabbit polyclonal to APAF1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human APAF1 aa 12-28 (N terminal).
    Sequence:

    HREALEKDIKTSYIMDHC


    (Peptide available as ab7873)

  • Positive control
    • HeLa whole cell lysate. This antibody gave a positive result in IF/ICC when used in the following formaldehyde fixed cell lines: HeLa.
  • General notes
    Apoptosis Protease Activating Factor.

Properties

Applications

Our Abpromise guarantee covers the use of ab2001 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 Use a concentration of 1 µg/ml.
ICC/IF Use a concentration of 10 µg/ml.
WB 1/1000 - 1/2000. Detects a band of approximately 141 kDa.Can be blocked with APAF1 peptide (ab7873).

Target

  • Function
    Oligomeric Apaf-1 mediates the cytochrome c-dependent autocatalytic activation of pro-caspase-9 (Apaf-3), leading to the activation of caspase-3 and apoptosis. This activation requires ATP. Isoform 6 is less effective in inducing apoptosis.
  • Tissue specificity
    Ubiquitous. Highest levels of expression in adult spleen and peripheral blood leukocytes, and in fetal brain, kidney and lung. Isoform 1 is expressed in heart, kidney and liver.
  • Sequence similarities
    Contains 1 CARD domain.
    Contains 1 NB-ARC domain.
    Contains 13 WD repeats.
  • Domain
    The CARD domain mediates interaction with APIP.
  • Cellular localization
    Cytoplasm.
  • Information by UniProt
  • Database links
  • Alternative names
    • APAF 1 antibody
    • Apaf-1 antibody
    • APAF_HUMAN antibody
    • Apaf1 antibody
    • Apoptotic peptidase activating factor 1 antibody
    • Apoptotic protease activating factor 1 antibody
    • Apoptotic protease activating factor antibody
    • Apoptotic protease-activating factor 1 antibody
    • CED 4 antibody
    • CED4 antibody
    • KIAA0413 antibody
    see all

Images

  • All lanes : Anti-APAF1 antibody (ab2001) at 1 µg/ml

    Lane 1 : K562 cell lysate in absence of blocking peptide
    Lane 2 : K562 cell lysate in presence of blocking peptide.

    Observed band size: 141 kDa
    why is the actual band size different from the predicted?

  • ab2001 at 1µg/ml staining Apaf1 in human heart tissue by IHC 
  • ICC/IF image of ab2001 stained HeLa cells. The cells were 4% formaldehyde 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 ab2001 at 10µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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:
  • You S  et al. Tunicamycin inhibits colon carcinoma growth and aggressiveness via modulation of the ERK-JNK-mediated AKT/mTOR signaling pathway. Mol Med Rep 17:4203-4212 (2018). Read more (PubMed: 29344654) »
  • Wang Q  et al. Resistant starch prevents tumorigenesis of dimethylhydrazine-induced colon tumors via regulation of an ER stress-mediated mitochondrial apoptosis pathway. Int J Mol Med 41:1887-1898 (2018). Read more (PubMed: 29393371) »
See all 8 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Chinese Hamster Cell lysate - whole cell (Chinese hamster ovary cell)
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris)
Loading amount
100000 cells
Treatment
100 nM Bortezomib for 16 hours
Specification
Chinese hamster ovary cell
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 09 2016

Application
Western blot
Sample
African Green Monkey Cell lysate - whole cell (COS-7 cells)
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris)
Loading amount
100000 cells
Treatment
30 nM Bortezomib for 24 hours
Specification
COS-7 cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 29 2015

Answer

Thank you for your reply.

As requested, I have placed a free of charge vial of ab59388 onto order 1083546. This should be shipped today or tomorrow and estimated delivery time is next Wednesday or Thursday.

I hope this will be helpful to you. If you need anything further, please do not hesitate to contact me. I am away from the office next week, but my colleagues will be happy to help you.

Read More

Answer

Thank you for your message and for kindly confirming these details.

According to our records, ab59388 was ordered in October which is past our 6 month guarantee period. However, reviewing the details, I can make an exception and provide a replacement or credit note for ab59388 in this case.

With regards to the other free of charge order for ab2001, order ref 1083546, I apologise for the delay. I would appreciate if you could confirm if you would like the replacement for ab59388 aswell, then I can add this to the same order and arrange for this to be shipped tomorrow (Wednesday) and it will take about 6 days to get to you.

I am sorry we do not ship antibodies on dry ice in order to avoid an extra freeze thaw cycle. They are shipped on ice, and with careful monitoring of customer feedback, we are not aware that this causes any problems with product quality.

Thank you for your time and cooperation. I look forward to hearing from you.

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

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:

Order number: 1083546

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

Answer

Thank you for your message and for kindly providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or credit note in compensation.

I look forward to hearing from you with details of how you would like to proceed.

Read More

Answer

Thank you for providing this further information.

I appreciate your cooperation and understand your concerns. It is regrettable the results have not been succesful. I have traced your previous corresopndence and reveiwed this case.

With regards to the band sizes expected for PAF1 protein in WB, the Uniprot protein database lists 6 isoforms of different sizes:

http://www.uniprot.org/uniprot/O14727
Isform 1 142 kDa
Isoform 2 141 Kda
Isoform 3 136 Kda
Isoform 4 137 kDa
Isoform 5 133 kDa
Isoform 6 37 kDa

Unfortunately, none of these would match the bands at 55 - 75 kDa that you are seeing. I can confirm this antibody has not specifically been tested on brain samples in WB and regrettably, I am having difficulty confirming which isoforms are known to be expressed in the brain with a literature search. I could just find the following article: http://www.pnas.org/content/104/52/20820.full that mentions normal brain has little Apaf-1 and tumors have a higher level, but unfortunately nothing specific to which isoforms are in the brain.

Could you confirm if any samples from other tissue have been included as a positive control? I can suggest this would be highly beneficial in this case.

In order to help with our investigation of this case before deciding how to proceed, I would appreciate if you are able to provide more detail as requested in the previous email. This information will also be helpful to our quality monitoring.

1. Please confirm the Abcam order reference number and date of purchase

2. For the secondary antibody, I can suggest an anti-rabbit secondary would be required to detect the rabbit polyclonal primary. An anti-goat would not detect rabbit polyclonal. If the correct secondary is being used, I can sugest it is also worth considering if the current vial is working well with other primary antibodies?.

3. Try a higher concentration of antibody to increase the signal. Try 1:1000. Incubate overnight 4oC.

4. Could you confirm which lysis buffer was used? I can recommend to lyse the cells in RIPA lysis buffer if this has not already been tried, before adding SDS loading buffer such as Laemmli to reduce and denature the samples. This should provide a more suitable protein preparation.

I would appreciate if you could also provide an image, including molecular weight markers, which would help us to assess the results.

I will be pleased to provide a free of charge replacmenet or credit note if the antibody is not working with the suggestions provided.

Thank you for your time and cooperation. We look forward to receiving the requested information and results from the positive control.

Read More

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 this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab2001 is tested and covered by our 6 month guarantee for use in WB and rat samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab2001. I would also appreciate if you can confirm some further details:

1. Please confirm the Abcam order reference number and date of purchase

2. For the secondary antibody, I can suggest an anti-rabbit secondary would be required to detect the rabbit polyclonal primary. An anti-goat would not detect rabbit polyclonal. If the correct secondary is being used, I can sugest it is also worth considering if the current vial is working well with other primary antibodies?.

3. Try a higher concentration of antibody to increase the signal. Try 1:1000. Incubate overnight 4oC.

4. Could you confirm which lysis buffer was used? I can recommend to lyse the cells in RIPA lysis buffer if this has not already been tried, before adding SDS loading buffer such as Laemmli to reduce and denature the samples. This should provide a more suitable protein preparation.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

Answer

Thank you again for your enquiry. Regarding ab4969, ab4957, and ab4958 (the peptides corresponding to the antibodies you indicated), they are all greater than 95% pure, and they were purified via HPLC. If you have any further questions, please contact us again.

Read More

Answer

Thank you for your enquiry. For ab2001 and ab7346, the peptides are non-HPLC, which averages out to 60-70% purity. Ab2001 (the antibody) was immunoaffinity chromatography purified IgG, and ab7346 (CCR5 antibody) is purified IgG. I will have to get back to you regarding the other peptides. If you have any additional questions, please let us know.

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

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