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AKT1/2/3 Recombinant Rabbit Monoclonal Antibody [JE75-09]

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Catalog# HA721870

AKT1/2/3 Recombinant Rabbit Monoclonal Antibody [JE75-09]

Application

  • WB

  • IF-Cell

  • FC

  • IP

Reactivity

  • Human

  • Mouse

  • Rat

  • Monkey

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

概要

製品名

AKT1/2/3 Recombinant Rabbit Monoclonal Antibody [JE75-09]

抗体の種類

Recombinant Rabbit monoclonal Antibody

免疫原

Recombinant protein within Human AKT1/2/3 aa 231-480 / 480.

種属反応性

Human, Mouse, Rat, Monkey

検証された応用例

WB, IF-Cell, FC, IP

分子量

Predicted band size: 56 kDa

陽性対照

HeLa cell lysate, MCF7 cell lysate, A549 cell lysate, U-2 OS cell lysate, NIH/3T3 cell lysate, MCF7, COS-1 cell lysate, RAW264.7 cell lysate, C6 cell lysate, PC-12 cell lysate, Mouse brain tissue lysate, Mouse heart tissue lysate, Mouse testis tissue lysate, Rat brain tissue lysate, Rat heart tissue lysate, Rat testis tissue lysate, RAW264.7, C6.

結合

unconjugated

クローン番号

JE75-09

RRID

AB_3096525

製品の特徴

形態

Liquid

濃度

保存方法

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

保存用バッファー

PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

アイソタイプ

IgG

精製方法

Protein A affinity purified.

応用希釈度

  • WB

  • 1:2,000

  • IF-Cell

  • 1:100

  • FC

  • 1:1,000

  • IP

  • 1-2μg/sample

ターゲット

機能

Akt, also referred to as PKB or Rac, plays a critical role in controlling cell survival and apoptosis. This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase. Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 . Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad, forkhead transcription factors, c-Raf, and caspase-9. PTEN phosphatase is a major negative regulator of the PI3K/Akt signaling pathway. LY294002 is a specific PI3 kinase inhibitor. Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β. Akt may also play a role in insulin stimulation of glucose transport. In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 and by negatively regulating the cyclin-dependent kinase inhibitors p27 Kip1 and p21 Waf1/Cip1. Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor. More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex.

背景文献

1. Tian X et al. Costunolide is a dual inhibitor of MEK1 and AKT1/2 that overcomes osimertinib resistance in lung cancer. Mol Cancer. 2022 Oct

2. Chen Z et al. Nuclear DEK preserves hematopoietic stem cells potential via NCoR1/HDAC3-Akt1/2-mTOR axis. J Exp Med. 2021 May

亜細胞局在

Cytoplasm, Nucleus, Cell membrane.

UNIPROT #

SwissProt: P31749 Human

SwissProt: P31751 Human

SwissProt: Q9Y243 Human

SwissProt: P31750 Mouse

SwissProt: Q60823 Mouse

SwissProt: Q9WUA6 Mouse

SwissProt: P47196 Rat

SwissProt: P47197 Rat

SwissProt: Q63484 Rat

別名

AKT antibody

AKT1 antibody

AKT1 kinase antibody

AKT1m antibody

AKT2 antibody

AKT2 kinase antibody

Akt3 antibody

AKT3_HUMAN antibody

CAKT antibody

CWS6 antibody

展開

画像

  • Western blot analysis of AKT1/2/3 on different lysates with Rabbit anti-AKT1/2/3 antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.<br /><br />Lane 1: HeLa cell lysate<br />Lane 2: MCF7 cell lysate<br />Lane 3: A549 cell lysate<br />Lane 4: U-2 OS cell lysate<br />Lane 5: NIH/3T3 cell lysate<br /><br />Lysates/proteins at 15 µg/Lane.<br /><br />Predicted band size: 56 kDa<br />Observed band size: 56 kDa<br /><br />Exposure time: 30 seconds; ECL: K1802;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of AKT1/2/3 on different lysates with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.

    Lane 1: HeLa cell lysate
    Lane 2: MCF7 cell lysate
    Lane 3: A549 cell lysate
    Lane 4: U-2 OS cell lysate
    Lane 5: NIH/3T3 cell lysate

    Lysates/proteins at 15 µg/Lane.

    Predicted band size: 56 kDa
    Observed band size: 56 kDa

    Exposure time: 30 seconds; ECL: K1802;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721870) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunocytochemistry analysis of MCF7 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AKT1/2/3 antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of MCF7 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Western blot analysis of AKT1/2/3 on different lysates with Rabbit anti-AKT1/2/3 antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/2,000 dilution.<br /><br />Lane 1: MCF7 cell lysate<br />Lane 2: A549 cell lysate<br />Lane 3: U-2 OS cell lysate<br />Lane 4: COS-1 cell lysate<br />Lane 5: NIH/3T3 cell lysate<br />Lane 6: RAW264.7 cell lysate<br />Lane 7: C6 cell lysate<br />Lane 8: PC-12 cell lysate<br />Lane 9: Mouse brain tissue lysate<br />Lane 10: Mouse heart tissue lysate<br />Lane 11: Mouse testis tissue lysate<br />Lane 12: Rat brain tissue lysate<br />Lane 13: Rat heart tissue lysate<br />Lane 14: Rat testis tissue lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 56 kDa<br />Observed band size: 56 kDa<br /><br />Exposure time: 2 minutes; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of AKT1/2/3 on different lysates with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/2,000 dilution.

    Lane 1: MCF7 cell lysate
    Lane 2: A549 cell lysate
    Lane 3: U-2 OS cell lysate
    Lane 4: COS-1 cell lysate
    Lane 5: NIH/3T3 cell lysate
    Lane 6: RAW264.7 cell lysate
    Lane 7: C6 cell lysate
    Lane 8: PC-12 cell lysate
    Lane 9: Mouse brain tissue lysate
    Lane 10: Mouse heart tissue lysate
    Lane 11: Mouse testis tissue lysate
    Lane 12: Rat brain tissue lysate
    Lane 13: Rat heart tissue lysate
    Lane 14: Rat testis tissue lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 56 kDa
    Observed band size: 56 kDa

    Exposure time: 2 minutes; ECL: K1801;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721870) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • AKT1/2/3 was immunoprecipitated from 0.2 mg MCF7 cell lysate with <a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a> at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using <a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a> at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.<br /><br />Lane 1: MCF7 cell lysate (input)<br />Lane 2: <a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a> IP in MCF7 cell lysate<br />Lane 3: Rabbit IgG instead of <a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a> in MCF7 cell lysate<br /><br />Blocking/Dilution buffer: 5% NFDM/TBST<br />Exposure time: 6 seconds; ECL: K1801

    AKT1/2/3 was immunoprecipitated from 0.2 mg MCF7 cell lysate with HA721870 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA721870 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

    Lane 1: MCF7 cell lysate (input)
    Lane 2: HA721870 IP in MCF7 cell lysate
    Lane 3: Rabbit IgG instead of HA721870 in MCF7 cell lysate

    Blocking/Dilution buffer: 5% NFDM/TBST
    Exposure time: 6 seconds; ECL: K1801

  • Immunocytochemistry analysis of RAW264.7 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AKT1/2/3 antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of RAW264.7 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Immunocytochemistry analysis of C6 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AKT1/2/3 antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of C6 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Flow cytometric analysis of C6 cells labeling AKT1/2/3.<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor&trade; 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (<a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of C6 cells labeling AKT1/2/3.

    Cells were fixed and permeabilized. Then stained with the primary antibody (HA721870, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

  • Flow cytometric analysis of RAW264.7 cells labeling AKT1/2/3.<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor&trade; 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (<a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of RAW264.7 cells labeling AKT1/2/3.

    Cells were fixed and permeabilized. Then stained with the primary antibody (HA721870, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

  • <span style="font-weight: bold;">☑ Knockdown (KD)</span><br /><br />Western blot analysis of AKT1/2/3 on different lysates with Rabbit anti-AKT1/2/3 antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/2,000 dilution.<br /><br />Lane 1: MCF7-si NT cell lysate<br />Lane 2: MCF7-si AKT1/2/3 cell lysate<br /><br />Lysates/proteins at 10 µg/Lane.<br /><br />Predicted band size: 56 kDa<br />Observed band size: 56 kDa<br /><br />Exposure time: 7 seconds; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA721870" style="font-weight: bold;text-decoration: underline;">HA721870</a>) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Knockdown (KD)

    Western blot analysis of AKT1/2/3 on different lysates with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/2,000 dilution.

    Lane 1: MCF7-si NT cell lysate
    Lane 2: MCF7-si AKT1/2/3 cell lysate

    Lysates/proteins at 10 µg/Lane.

    Predicted band size: 56 kDa
    Observed band size: 56 kDa

    Exposure time: 7 seconds; ECL: K1801;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721870) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

ご注意ください: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

論文での実績

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