Anti-APAF1抗体(ab2001)

Reviews (3)Q&A (8)References (18)

Key features and details

  • Rabbit polyclonal to APAF1
  • Suitable for: IHC-P, ICC/IF, WB
  • Reacts with: Mouse, Rat, Human
  • Isotype: IgG

概述

  • 产品名称

    Anti-APAF1抗体
    参阅全部 APAF1 一抗

  • 描述

    兔多克隆抗体to APAF1

  • 宿主

    Rabbit

  • 经测试应用

    适用于: IHC-P, ICC/IF, WBmore details

  • 种属反应性

    与反应: Mouse, Rat, Human

  • 免疫原

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

    HREALEKDIKTSYIMDHC


    (Peptide available as ab7873)

    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI

  • 阳性对照

    • HeLa whole cell lysate. This antibody gave a positive result in IF/ICC when used in the following formaldehyde fixed cell lines: HeLa.

  • 常规说明

    Apoptosis Protease Activating Factor.

性能

应用

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.

应用 Ab评论 说明
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.

靶标

  • 功能

    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.

  • 组织特异性

    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.

  • 序列相似性

    Contains 1 CARD domain.
    Contains 1 NB-ARC domain.
    Contains 13 WD repeats.

  • 结构域

    The CARD domain mediates interaction with APIP.

  • 细胞定位

    Cytoplasm.
  • Information by UniProt

  • 数据库链接

    see all

  • 别名

    • 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

图片

  • Western blot - Anti-APAF1 antibody (ab2001)
    Western blot - Anti-APAF1 antibody (ab2001)
    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?

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-APAF1 antibody (ab2001)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-APAF1 antibody (ab2001)
    ab2001 at 1µg/ml staining Apaf1 in human heart tissue by IHC 
  • Immunocytochemistry/ Immunofluorescence - Anti-APAF1 antibody (ab2001)
    Immunocytochemistry/ Immunofluorescence - Anti-APAF1 antibody (ab2001)

    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.

文献

发表研究结果有使用 ab2001？请让我们知道，以便我们可以引用本数据表中的参考文章。

ab2001 被引用在 18 文献中.

  • Miao J  et al. Hydroxytyrosol protects against myocardial ischemia reperfusion injury by inhibiting mitochondrial permeability transition pore opening. Exp Ther Med 17:671-678 (2019). PubMed: 30651849
  • Fang H  et al. miR-937 regulates the proliferation and apoptosis via targeting APAF1 in breast cancer. Onco Targets Ther 12:5687-5699 (2019). PubMed: 31410016
  • Wu B  et al. Combination of Sodium Selenite and Doxorubicin Prodrug Ac-Phe-Lys-PABC-ADM Affects Gastric Cancer Cell Apoptosis in Xenografted Mice. Biomed Res Int 2019:2486783 (2019). PubMed: 31531348
  • Zou L  et al. Bone marrow mesenchymal stem cell-derived exosomes protect against myocardial infarction by promoting autophagy. Exp Ther Med 18:2574-2582 (2019). PubMed: 31555366
  • Wang W  et al. miR-222 induces apoptosis in human intervertebral disc nucleus pulposus cells by targeting Bcl-2. Mol Med Rep 20:4875-4882 (2019). PubMed: 31638197
View all Publications for this product

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1-10 of 11 Abreviews or Q&A

Western blot abreview for Anti-APAF1 antibody

 Good
Abreviews
abreview image
Application
Western blot
Sample
Rat Cell lysate - whole cell (primary cardiac fibroblasts)
Gel Running Conditions
Reduced Denaturing (8.5%)
Loading amount
20 µg
Specification
primary cardiac fibroblasts
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 25°C
Read More

Abcam user community

Verified customer

提交于 Jun 11 2019

Western blot abreview for Anti-APAF1 antibody

 Excellent
Abreviews
abreview image
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
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Abcam user community

Verified customer

提交于 Feb 09 2016

Western blot abreview for Anti-APAF1 antibody

 Excellent
Abreviews
abreview image
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
Read More

Abcam user community

Verified customer

提交于 Jun 29 2015

Question

Dear Cate,
Thank you for your reply from mailto:technical@abcam.com. I would like to get the replacement offer for ab59388 along with ab2001. I hope you can ship both the items together.
Best,

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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.

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Question

Hi,
Thanks for your reply. I have used the same type of samples ( of course different preparation) which I have used for ab2001. I have tried in male Wistar rat brain homogenate and PC12 cell lysate as well. I have used it for western blot analysis, with caspse 3, I tried dilutions up to 1:500. Blocking with BSA, 4 degree incubation with primary antibody. I could not get signals with any of the variations. My western is working well as I am getting signals for house keeping genes in the same blot. If I have to provide further details please let me know.
Best,

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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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Question

Thank you for your suggestion and co-operation. I would appreciate if you kindly replace the antibody so that I can carry out my experiment. I look forward for your reply,
Best,

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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.

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Question

I have tried your following suggestions, correct secondary antibody is used. the vial is correctly working with other primary antibody. I can send you the DAB stained picture,let me know whether it will help you.
best,

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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.

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Question

Product code: 2001
Lot number: GR 26336-2
Inquiry: Hello, I had contacted the technical help desk earlier for my problem with immunoblot. I tried the suggestions and I got bands, in one blot i gt band around 130 kDa, with some non specific bands but in subsequent blot i got single band around (range between 55-75 kDa). I checked with the literature and i got variation in band size of Apaf1. Please provide me information of the variation of Apaf 1 size. I am looking for Apaf1 in apoptotic brain tissues. your help would be highly appreciated. best,

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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.

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Question

LOT NUMBER GR 26336-2
ORDER NUMBER -- NOT SPECIFIED --
DESCRIPTION OF THE PROBLEM No signal
SAMPLE CYTOSOLIC FRACTION OF MALE WISTER RAT BRAIN HOMOGENATE- , PC12-LYSIS PROTEIN, NEURA 2A LYSED PROTEIN
PRIMARY ANTIBODY 1:2000
DILUTION IN 0.03%BSA IN TBST
DETECTION METHOD ecl
POSITIVE AND NEGATIVE CONTROLS USED no positive control
ANTIBODY STORAGE CONDITIONS -80c
SAMPLE PREPARATION DID HEATING AT 94 DEGREES FOR 4 MINUTES WITH DYE
AMOUNT OF PROTEIN LOADED 100 microgram
ELECTROPHORESIS/GEL CONDITIONS 8% gel
TRANSFER AND BLOCKING CONDITIONS BSA BUFFER, OVERNIGHT
SECONDARY ANTIBODY ANTI GOAT ANTIBODY 1:10000 DILUTION
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5
HAVE YOU RUN A "NO PRIMARY" CONTROL? No
WHAT STEPS HAVE YOU ALTERED? incubation PERIOD of primary antibody increased, 4 C, RT tried

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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 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.

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Question

This email is additional information for my previous email whichi subject is "Question about antibody and peptid". I forgot to ask you how pure they are and which method these peptides have been purified by. Thank you for your cooperation. ab2001, HREALEKDIKTSYIMDHC ab7346, SSPIYDINYYTSEPC ab3430, MSDFDSNPFADPDLNNPC ab3346, MSAPAQPPAEGTEGAAPGC ab3347, MSATAATVPPAAPAGEGGC Thank you for your cooperation

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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.

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1-10 of 11 Abreviews or Q&A

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