Anti-PHAPI2 / APRIL 抗体 (ab4224)

Submit a review Q&A (7)References (3)

Key features and details

  • Goat polyclonal to PHAPI2 / APRIL
  • Suitable for: IHC-P, WB
  • Reacts with: Mouse, Rat, Human, Pig
  • Isotype: IgG

製品の概要

  • 製品名

    Anti-PHAPI2 / APRIL antibody
    PHAPI2 / APRIL 一次抗体 製品一覧

  • 製品の詳細

    Goat polyclonal to PHAPI2 / APRIL

  • 由来種

    Goat

  • 特異性

    This antibody does NOT recognize the TNF family member also known as APRIL - LocusLink Number 8741

  • アプリケーション

    適用あり: IHC-P, WBmore details

  • 種交差性

    交差種: Mouse, Rat, Human, Pig
    交差が予測される動物種: Cow, Dog

  • 免疫原

    Synthetic peptide: KRKRETDDEGEDD, corresponding to C terminal amino acids 237-249 of Human PHAPI2/ APRIL.

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

  • ポジティブ･コントロール

    • Human Tonsil lysate.

  • 特記事項

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab4224 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
IHC-P Use a concentration of 3 - 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
WB Use a concentration of 0.1 - 0.3 µg/ml. Detects a band of approximately 30 kDa (predicted molecular weight: 29 kDa).

1 hour primary incubation is recommended for this product.

ターゲット情報

  • 機能

    Multifunctional protein working as a cell cycle progression factor as well as a cell survival factor. Required for the progression from the G1 to the S phase. Anti-apoptotic protein which functions as a caspase-3 inhibitor. Has no phosphatase 2A (PP2A) inhibitor activity (By similarity). Exhibits histone chaperone properties, stimulating core histones to assemble into a nucleosome.

  • 組織特異性

    Expressed in heart, lung, pancreas, prostate and in spleen, thymus and placenta.

  • 配列類似性

    Belongs to the ANP32 family.
    Contains 4 LRR (leucine-rich) repeats.
    Contains 1 LRRCT domain.

  • ドメイン

    Histone binding is mediated by the concave surface of the LRR region.

  • 翻訳後修飾

    Some glutamate residues are glycylated by TTLL8. This modification occurs exclusively on glutamate residues and results in a glycine chain on the gamma-carboxyl group.

  • 細胞内局在

    Nucleus. Accumulates in the nuclei at the S phase and Cytoplasm. Lacks a nuclear localization signal.
  • Information by UniProt

  • 参照データベース

    see all

  • 別名

    • Acidic (leucine rich) nuclear phosphoprotein 32 family member B antibody
    • Acidic leucine rich nuclear phosphoprotein 32 family member B antibody
    • Acidic leucine-rich nuclear phosphoprotein 32 family member B antibody
    • Acidic nuclear phosphoprotein 32 family member B antibody
    • Acidic protein rich in leucines antibody
    • AN32B_HUMAN antibody
    • ANP 32B antibody
    • ANP32B antibody
    • APRIL antibody
    • OTTHUMP0000006377 antibody
    • PAL 31 antibody
    • PAL31 antibody
    • PHAPI 2 antibody
    • PHAPI2 antibody
    • PHAPI2 protein antibody
    • PHAPI2a antibody
    • Proliferation related acidic leucine rich protein antibody
    • Putative HLA-DR-associated protein I-2 antibody
    • Silver stainable protein SSP 29 antibody
    • Silver stainable protein SSP29 antibody
    • Silver-stainable protein SSP29 antibody
    • Ssp 29 antibody
    • Ssp29 antibody
    see all

画像

  • Western blot - Anti-PHAPI2 / APRIL antibody (ab4224)
    Western blot - Anti-PHAPI2 / APRIL antibody (ab4224)
    All lanes : Anti-PHAPI2 / APRIL antibody (ab4224) at 0.1 µg/ml

    Lane 1 : Human tonsil lysate
    Lane 2 : Rat spleen lysate
    Lane 3 : Pig spleen lysate

    Lysates/proteins at 35 mg/ml per lane.

    Predicted band size: 29 kDa
    Observed band size: 28-30 kDa
    why is the actual band size different from the predicted?



    Primary incubation was 1 hour. Detected by chemiluminescence.

  • Western blot - Anti-PHAPI2 / APRIL antibody (ab4224)
    Western blot - Anti-PHAPI2 / APRIL antibody (ab4224)
    Anti-PHAPI2 / APRIL antibody (ab4224) at 0.1 µg/ml + NIH3T3 cell lysate at 35 µg

    Predicted band size: 29 kDa
    Observed band size: 30 kDa why is the actual band size different from the predicted?



    Primary incubation was 1 hour. Detected by chemiluminescence.

  • Western blot - Anti-PHAPI2 / APRIL antibody (ab4224)
    Western blot - Anti-PHAPI2 / APRIL antibody (ab4224)
    ab4224 at a concentration of 1 µg/ml, staining ~30 kDa PHAP I2 in human lymph node lysate (RIPA bufer, 30 µg total proteion per lane) by western blot (primary incubated for 1 hour, detected by ECL). ab4224 at a concentration of 1 µg/ml, staining ~30 kDa PHAP I2 in human lymph node lysate (RIPA bufer, 30 µg total proteion per lane) by western blot (primary incubated for 1 hour, detected by ECL).
  • Western blot - Anti-PHAPI2 / APRIL antibody (ab4224)
    Western blot - Anti-PHAPI2 / APRIL antibody (ab4224)
    Anti-PHAPI2 / APRIL antibody (ab4224) at 0.3 µg/ml + A431 lysate in RIPA buffer at 35 µg

    Developed using the ECL technique.

    Predicted band size: 29 kDa
    Observed band size: 29 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHAPI2 / APRIL antibody (ab4224)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHAPI2 / APRIL antibody (ab4224)

    ab4224 (3.8µg/ml) staining of paraffin embedded Human Breast tissue following Steamed antigen retrieval with citrate buffer pH 6 and AP-staining shows nuclear staining in lobular cells.

参考文献 (3)

ab4224 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab4224 は 3 報の論文で使用されています。

  • Jamison JT  et al. Organelles do not colocalize with mRNA granules in post-ischemic neurons. Neuroscience 199:394-400 (2011). PubMed: 21978884
  • Khan AP  et al. Quantitative proteomic profiling of prostate cancer reveals a role for miR-128 in prostate cancer. Mol Cell Proteomics 9:298-312 (2010). WB ; Human . PubMed: 19955085
  • Munemasa Y  et al. Promoter region-specific histone incorporation by the novel histone chaperone ANP32B and DNA-binding factor KLF5. Mol Cell Biol 28:1171-81 (2008). WB ; Human . PubMed: 18039846

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1-7 of 7 Abreviews or Q&A

Question

For GST-APRIL, 1:1000 dilution of ab4224 works fine. We used HeLa cell lysates and we see a faint band with 1:50 dilution of the ab4224.

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Answer

Thanks for your email. What size band did you see with the GST fusion protein? As I mentioned, we do recommend using human lymph node lysate as a positive control with this antibody. To our knowledge it has not been tested with HeLa cells, I don't know what the expression level is in that cell line.

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Question

Thanks for your response. We started with the recommended dilution of 1:500 and came down to 1:50 at which a very faint band is detected in human cell lines. Our secondary antibodies are working fine. We are doing numerous western these days. GST-APRIL does work fine with ab4224. Thanks

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Answer

Thank you for your email. At what dilutions of ab4224 did you see a signal with the GST-APRIL purified protein? Ab4224 was characterized using Human Lymph Node lysate, which is recommended as a positive control. You didn't specifically mention which human lysates you used, so I'm not sure if you tried that or not.

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Question

BATCH NUMBER 84574 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal. SAMPLE Protein lysates from human, mouse, rat cells. With human lysates we see a very very weak signal with antibody dilution of 1:50 incubating overnight. With others we don't see anything. PRIMARY ANTIBODY Anti-PHAPI2/ abcam SECONDARY ANTIBODY From Santa Cruz DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED WE used GST-APRIL purified protein that is detected as a nice band. ANTIBODY STORAGE CONDITIONS Stored at -20 in aliquots SAMPLE PREPARATION With PI. Other western blots with other antibodies don't have any problems. AMOUNT OF PROTEIN LOADED Normally we load 30 ug, but for this antibody we have even loaded 100ug protein on a small gel. ELECTROPHORESIS/GEL CONDITIONS 10 % denaturing gel. TRANSFER AND BLOCKING CONDITIONS Wet transfer overnight on nitrocellulose. Blocking with 5% milk. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? >5 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

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Answer

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab4224. I do need a few more details from you in order to investigate this problem. What dilutions of ab4224 have you tried? Is your secondary antibody working properly with other primaries? Also, you mentioned that you "used GST-APRIL purified protein that is detected as a nice band." Did this work then with ab4224? Thanks again, and I look forward to hearing from you.

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Question

Thank you for your quick reply. We received an inquire about #ab4224 (Lot#51775) from our customer. They would like to know that you have performed QC that Lot#51775 is able to used to WB. They would like you to send them, if you have the certification about it. Please advise them it.

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Answer

Thank you for your enquiry. We are still selling the first batch - all data on the datasheet is therefore correct.

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Question

I am sorry that I have not written to you about the matter. The customer used replaced item, and he got good result. The problem was solved. Please do not worry.

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Answer

Thank you for this feedback information!

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Question

We received an inquiry from the customer who has used your product #ab4224 (Anti APRIL), Lot#17967. He tried to detect (1) and (2) using whole cell lysate by western blot. (1) full length APRIL-Myc tag fusion protein overexpressed in HeLa cell (2) endogenous APRIL in HeLa cell But he could not detect any signal and highly background was seen. Here is his condition. primary antibody: #ab4224, Lot17967, dilute 1:500 (1ug/ml), 90min at Room Temperature secondary antibody: MBL #274 (Anti goat IgG-biotin), dilute 1:500 He could detect (2) (Myc fusion protein) by Anti-Myc tag antibody. So he thougnt his assay protocol did not have any problem. Could you advise him and send us the replacement?

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Answer

We are very sorry but we can't provide more technical support in this matter. This antibody was produced by another company and the information we have is very limited. We have do not have other vials from a different batch, so either we can offer your customer a replacement vial from the same batch or a refund. Please do let us know what your customer would prefer to get.

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Question

I purchased your anti PHAPI2 antibody (cat. 4224-100, lot. 17967). After several trials I was not able to visualize the protein in WB, even using the antibody at 1.0 micrograms/microliter. I used a whole cell lysate of 293 cells transfected with an HA-PHAPI2 construct. I would like to ask you whether it would be possible any product exchange. In alternative it would be nice to have a small amount of the same antibody (possibly from another lot) to use fo a further test. In your reply you can find the answers to the questions that you made me. I hope they would be useful to understand the nature of the problem. I'll be happy to answer to more questions if needed. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). High diffuse Background, no specific bands (neither endogenous nor transfected) 2. On what material are you testing the antibody in WB? 293T cells transfected with a recombinant HA-PHAPI2 construct Species? Human Cell extract/ Nuclear extract? Cell extract Purified protein? NO Recombinant protein?NO 3. How much protein did you load? about 40 micrograms How did you prepare the lysate for the analysis (protease inhibitors etc)? Laemli Buffer 1X Did you heat the samples?Yes, 5 min. @95 Celsisus degrees 4. Primary Antibody Specification (in which species was it raised against)? Goat At what dilution(s) have you tested this antibody?1.0 microgram/milliliter (1:500) Incubation time, wash step? 1hr @room temperature (also o/n @4 degrees), 3 washes 10 minutes each 5. Secondary Antibody Specification (in which species was it raised against)?Rabbit At what dilution(s) have you tested this antibody?1:2000 Incubation time, wash step?1 hr @room temperature Do you know whether the problems you are experiencing come from the secondary? The secondary ab works fine with other goat primary antibodies 6. What detection method are you using? Amersham ECL or in alternative Pierce FemtoECL 7. Background bands No background bands Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a ?No primary? control) Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) At what size are the bands migrating? Could they be degradation products of your target? Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 8. Optimization attempts How many times have you tried the Western? Three times, changing the incubation times of the primary antibody Do you obtain the same results every time e.g. are background bands always in the same place? teins What steps have you altered? 9. Did you apply positive and negative controls along with the samples? Please specify. I transfected 293T cells in parallel with the following constructs: Mock, HA-PHAPI, HA-PHAPI2a. Checked for the presence of the proteins on the blot by probing with HA antibody, result: positive for both recombinant proteins. The sequence of the constructs was verified by sequencing.

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Answer

Thank you for your enquiry. We are very sorry to hear that you are having problem with this antibody. The question we would need to ask is whether the HA tag could be blocking the epitope ie is it on the C-terminus or the N-terminus of the fusion protein? We are looking forward to hearing from you soon.

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