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Cell Biology Apoptosis Intracellular Kinases
使用敲除细胞株进行验证重组RabMAb

重组Anti-PAK2抗体[EP796Y] (ab76293)

  • Datasheet
Reviews (2) Submit a question References (8)

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Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Immunocytochemistry/ Immunofluorescence - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Flow Cytometry - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Immunocytochemistry/ Immunofluorescence - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Flow Cytometry - Anti-PAK2 antibody [EP796Y] (ab76293)
  • Anti-PAK2 antibody [EP796Y] (ab76293)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP796Y] to PAK2
  • Suitable for: IP, ICC/IF, WB, IHC-P, Flow Cyt
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

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概述

  • 产品名称

    Anti-PAK2抗体[EP796Y]
    参阅全部 PAK2 一抗
  • 描述

    兔单克隆抗体[EP796Y] to PAK2
  • 宿主

    Rabbit
  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    IP
    Mouse
    Human
    WB
    Mouse
    Rat
    Human
    See all applications and species data
  • 免疫原

    Synthetic peptide within Human PAK2 aa 1-100 (N terminal). The exact sequence is proprietary.
    Database link: Q13177

  • 阳性对照

    • WB: HeLa, NIH/3T3, RAW 264.7 and C6 cell lysates. IHC-P; Human breast carcinoma tissue IF/ICC: T47D cell line. Flow Cyt: HeLa cells. IP: HeLa and NIH/3T3 cell lysates.
  • 常规说明

     

     

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

性能

  • 形式

    Liquid
  • 存放说明

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • 存储溶液

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • 纯度

    Protein A purified
  • 克隆

    单克隆
  • 克隆编号

    EP796Y
  • 同种型

    IgG
  • 研究领域

    • Cell Biology
    • Apoptosis
    • Intracellular
    • Kinases
    • Microbiology
    • Interspecies Interaction
    • Host Virus Interaction
    • Signal Transduction
    • Protein Phosphorylation
    • Ser / Thr Kinases
    • Other Kinases
    • Cancer
    • Signal transduction
    • Protein phosphorylation
    • Serine/threonine kinases
    • Other

相关产品

  • Alternative Versions

    • APC Anti-PAK2 antibody [EP796Y] (ab225022)
    • PE Anti-PAK2 antibody [EP796Y] (ab225023)
    • Alexa Fluor® 647 Anti-PAK2 antibody [EP796Y] (ab225024)
    • Anti-PAK2 antibody [EP796Y] - BSA and Azide free (ab227990)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human PAK2 knockout HeLa cell line (ab264814)
  • KO cell lysates

    • Human PAK2 knockout HeLa cell lysate (ab257573)
  • KO cell pellets

    • Human PAK2 knockout HeLa cell pellet (ab278854)
  • Recombinant Protein

    • Recombinant Human PAK2 protein (ab125597)

应用

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab76293 in the following tested applications.

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

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

应用 Species
Flow Cyt
Human
ICC/IF
Human
IHC-P
Human
IP
Mouse
Human
WB
Mouse
Rat
Human
应用 Ab评论 说明
IP
1/30.
ICC/IF (1)
1/100 - 1/250.
WB (1)
1/5000. Predicted molecular weight: 58 kDa.

For unpurified use at 1/1000 - 1/2000.

IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

This antibody may not be suitable for IHC with mouse or rat samples
Use of HRP conjugated or polymerized HRP secondary antibody is recommended. Stronger signals have been found using the polymerized HRP secondary.

Flow Cyt
1/20.

For unpurified use at 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

说明
IP
1/30.
ICC/IF
1/100 - 1/250.
WB
1/5000. Predicted molecular weight: 58 kDa.

For unpurified use at 1/1000 - 1/2000.

IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

This antibody may not be suitable for IHC with mouse or rat samples
Use of HRP conjugated or polymerized HRP secondary antibody is recommended. Stronger signals have been found using the polymerized HRP secondary.

Flow Cyt
1/20.

For unpurified use at 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

靶标

  • 功能

    The activated kinase acts on a variety of targets. Phosphorylates ribosomal protein S6, histone H4 and myelin basic protein. Full length PAK 2 stimulates cell survival and cell growth. The process is, at least in part, mediated by phosphorylation and inhibition of pro-apoptotic BAD. Caspase-activated PAK-2p34 is involved in cell death response, probably involving the JNK signaling pathway. Cleaved PAK-2p34 seems to have a higher activity than the CDC42-activated form.
  • 组织特异性

    Ubiquitously expressed. Higher levels seen in skeletal muscle, ovary, thymus and spleen.
  • 序列相似性

    Belongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. STE20 subfamily.
    Contains 1 CRIB domain.
    Contains 1 protein kinase domain.
  • 翻译后修饰

    Full length PAK 2 is autophosphorylated when activated by CDC42/p21. Following cleavage, both peptides, PAK-2p27 and PAK-2p34, become highly autophosphorylated, with PAK-2p27 being phosphorylated on serine and PAK-2p34 on threonine residues, respectively. Autophosphorylation of PAK-2p27 can occur in the absence of any effectors and is dependent on phosphorylation of Thr-402, because PAK-2p27 is acting as an exogenous substrate.
    During apoptosis proteolytically cleaved by caspase-3 or caspase-3-like proteases to yield active PAK-2p34.
    Ubiquitinated, leading to its proteasomal degradation.
    PAK-2p34 is myristoylated.
  • 细胞定位

    Cytoplasm and Nucleus. Cytoplasm > perinuclear region. Membrane. Interaction with ARHGAP10 probably changes PAK-2p34 location to cytoplasmic perinuclear region. Myristoylation changes PAK-2p34 location to the membrane.
  • Target information above from: UniProt accession Q13177 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • 数据库链接

    • Entrez Gene: 5062 Human
    • Entrez Gene: 224105 Mouse
    • Entrez Gene: 29432 Rat
    • GenBank: NP_002568.2 Human
    • Omim: 605022 Human
    • SwissProt: Q13177 Human
    • SwissProt: Q8CIN4 Mouse
    • SwissProt: Q64303 Rat
    • Unigene: 518530 Human
    • Unigene: 234204 Mouse
    • Unigene: 3840 Rat
    see all
  • 别名

    • C-t-PAK2 antibody
    • CB422 antibody
    • EC 2.7.11.1 antibody
    • Gamma PAK antibody
    • Gamma-PAK antibody
    • hPAK65 antibody
    • Kinase antibody
    • p21 (CDKN1A) activated kinase 2 antibody
    • p21 (CDKN1A)-activated kinase 2a antibody
    • p21 activated kinase 2 antibody
    • p21 protein (Cdc42/Rac)-activated kinase 2 antibody
    • p21 protein Cdc42 Rac activated kinase 2 antibody
    • p21-activated kinase 2 antibody
    • p21-activated kinase, 65-KD antibody
    • p21-activated protein kinase I antibody
    • p21CDKN1A activated kinase 2 antibody
    • p27 antibody
    • p34 antibody
    • p58 antibody
    • p65PAK antibody
    • PAK 2 antibody
    • PAK-2 antibody
    • PAK-2p34 antibody
    • Pak2 antibody
    • PAK2_HUMAN antibody
    • PAK65 antibody
    • PAKgamma antibody
    • S6 H4 kinase antibody
    • S6/H4 kinase antibody
    • Serine threonine protein kinase PAK 2 antibody
    • Serine/threonine protein kinase PAK 2 antibody
    see all

图片

  • Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
    Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
    All lanes : Anti-PAK2 antibody [EP796Y] (ab76293) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : PAK2 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 58 kDa
    Observed band size: 60 kDa
    why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab76293 observed at 60 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab76293 was shown to react with PAK2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264814 (knockout cell lysate ab257573) was used. Wild-type HeLa and PAK2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab76293 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (ab76293)
    Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (ab76293)
    PAK2 was immunoprecipitated from 0.35 mg NIH/3T3 (Mouse embryonic fibroblast) cell lysate 10 µg with ab76293 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab76293 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

    Lane 1: NIH/3T3 (Mouse embryonic fibroblast) cell lysate 10 µg

    Lane 2: ab76293 IP in NIH/3T3 cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab76293 in HeLa cell lysate

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 7 seconds

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK2 antibody [EP796Y] (ab76293)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK2 antibody [EP796Y] (ab76293)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling PAK2 with Purified ab76293 at 1:100 dilution (2.02 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

  • Immunocytochemistry/ Immunofluorescence - Anti-PAK2 antibody [EP796Y] (ab76293)
    Immunocytochemistry/ Immunofluorescence - Anti-PAK2 antibody [EP796Y] (ab76293)

    Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling PAK2 with purified ab76293 at 1:100 dilution (2.0μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • Flow Cytometry - Anti-PAK2 antibody [EP796Y] (ab76293)
    Flow Cytometry - Anti-PAK2 antibody [EP796Y] (ab76293)

    Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PAK2 with purified ab76293 at 1:20 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilized with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

  • Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
    Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)

    Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: PAK2 knockout HAP1 cell lysate (20 µg)
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: Human lymph node tissue lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab76293 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

    Unpurified ab76293 was shown to specifically react with PAK2 when PAK2 knockout samples were used. Wild-type and PAK2 knockout samples were subjected to SDS-PAGE. ab76293 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

  • Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
    Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
    All lanes : Anti-PAK2 antibody [EP796Y] (ab76293) at 1/5000 dilution (purified)

    Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
    Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
    Lane 3 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
    Lane 4 : C6 (Rat glial tumor glial cell) whole cell lysates

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

    Predicted band size: 58 kDa



    Blocking and diluting buffer: 5% NFDM/TBST

  • Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
    Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)
    Anti-PAK2 antibody [EP796Y] (ab76293) at 1/2000 dilution (unpurified) + HeLa cell lysate at 10 µg

    Secondary
    HRP labelled goat anti-rabbit at 1/2000 dilution

    Predicted band size: 58 kDa
    Observed band size: 61 kDa why is the actual band size different from the predicted?

  • Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (ab76293)
    Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (ab76293)
    PAK2 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) cell lysate 10 µg with ab76293 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab76293 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

    Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) cell lysate 10 µg

    Lane 2: ab76293 IP in HeLa cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab76293 in HeLa cell lysate

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 7 seconds

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK2 antibody [EP796Y] (ab76293)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK2 antibody [EP796Y] (ab76293)

    Unpurified ab76293, at a 1/100 dilution, staining PAK2 in paraffin embedded human breast carcinoma tissue by Immunohistochemistry.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Immunocytochemistry/ Immunofluorescence - Anti-PAK2 antibody [EP796Y] (ab76293)
    Immunocytochemistry/ Immunofluorescence - Anti-PAK2 antibody [EP796Y] (ab76293)

    ICC/IF image of unpurified ab76293 stained T47D cells. The cells were 100% methanol fixed (5 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 (ab76293, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.

  • Flow Cytometry - Anti-PAK2 antibody [EP796Y] (ab76293)
    Flow Cytometry - Anti-PAK2 antibody [EP796Y] (ab76293)

    Overlay histogram showing HeLa cells stained with unpurified ab76293 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76293, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

  • Anti-PAK2 antibody [EP796Y] (ab76293)
    Anti-PAK2 antibody [EP796Y] (ab76293)

实验方案

  • Flow cytometry protocols
  • Immunohistochemistry protocols
  • Immunocytochemistry & immunofluorescence protocols
  • Western blot protocols

Click here to view the general protocols

数据表及文件

    • Datasheet
  • 文献 (8)

    发表研究结果有使用 ab76293?请让我们知道,以便我们可以引用本数据表中的参考文章。

    ab76293 被引用在 8 文献中.

    • Yao GW  et al. P21 activated kinase 2 promotes pancreatic cancer growth and metastasis. Oncol Lett 17:3709-3718 (2019). PubMed: 30930982
    • Grebenová D  et al. PAK1, PAK1?15, and PAK2: similarities, differences and mutual interactions. Sci Rep 9:17171 (2019). PubMed: 31748572
    • Hu S  et al. MicroRNA-455-3p promotes TGF-ß signaling and inhibits osteoarthritis development by directly targeting PAK2. Exp Mol Med 51:1-13 (2019). PubMed: 31586040
    • Hiroyasu S  et al. Loss of ß-PIX inhibits focal adhesion disassembly and promotes keratinocyte motility via myosin light chain activation. J Cell Sci 130:2329-2343 (2017). PubMed: 28596238
    • Shao Y  et al. Human cytomegalovirus miR-US4-5p promotes apoptosis via downregulation of p21-activated kinase 2 in cultured cells. Mol Med Rep 16:4171-4178 (2017). PubMed: 28765936
    • Jiang N  et al. In vivo quantitative phosphoproteomic profiling identifies novel regulators of castration-resistant prostate cancer growth. Oncogene N/A:N/A (2014). Mouse . PubMed: 25065596
    • Chen G  et al. TGFß receptor I transactivation mediates stretch-induced Pak1 activation and CTGF upregulation in mesangial cells. J Cell Sci 126:3697-712 (2013). PubMed: 23781022
    • Langlois B  et al. LRP-1 promotes cancer cell invasion by supporting ERK and inhibiting JNK signaling pathways. PLoS One 5:e11584 (2010). WB . PubMed: 20644732

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    Immunocytochemistry/ Immunofluorescence abreview for Anti-PAK2 antibody [EP796Y]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Rat Cell (heart)
    Permeabilization
    Yes - 0.1% Triton
    Specification
    heart
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.5% · Temperature: 22°C
    Fixative
    Methanol
    Read More

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    提交于 Feb 27 2019

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    研究类别(A-Z)
    • 肿瘤研究
    • 心血管研究
    • 细胞生物学
    • 发育生物学
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