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AKIN10 | SNF1-related protein kinase catalytic subunit alpha KIN10

AS10 919 | Clonality: Polyclonal Host: Rabbit | Reactivity: Arabidopsis thaliana

AKIN10 | SNF1-related protein kinase catalytic subunit alpha KIN10 in the group Antibodies Plant/Algal  / Developmental Biology / Signal transduction at Agrisera AB (Antibodies for research) (AS10 919)
AKIN10 | SNF1-related protein kinase catalytic subunit alpha KIN10



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Product name, number (Agrisera, Sweden)

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Product Information

Immunogen

KLH-conjugated synthetic peptide derived from C-terminal part of Arabidopsis thaliana AKIN10 sequence UniProt: Q38997, TAIR: At3g01090

Host Rabbit
Clonality Polyclonal
Purity Immunogen affinity purified serum in PBS pH 7.4.
Format Lyophilized
Quantity 100 ĩg
Reconstitution For reconstitution add 50 ĩl/tube of sterile water
Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Tested applications Immunolocalization (IL), Western blot (WB)
Recommended dilution 1: 50 (IL), 1 : 500 (WB)
Expected | apparent MW 61,1 kDa

Reactivity

Confirmed reactivity Arabidopsis thaliana
Predicted reactivity Arabidopsis thaliana
Not reactive in

Oryza sativa, Solanum lycopersicum, Vitis vinifera

Application examples

Application examples Application example

western blot using anti-AKIN10 antibodies


Arabidopsis thaliana total proteins were extracted in Hepes buffer (50mM Hepes-NaOH pH 7.8; 2mM EDTA pH 8.0; 1mM DTT; Phosphatase and Protease inhibitor cocktails). After centrifugation, the supernatant was recovered and protein concentration determined using the Bradford protein assay. 5, 10, 20 and 30 μg of total protein were resolved, for each plant extract, by SDS-PAGE, transferred to a PVDF membrane and analyzed by immunoblotting with α-AKIN10 antibody (Agrisera; AS10 919; 1:500; 1% non-fat Milk in TBS O/N at 4°C). Secondary antibody anti-rabbit HRP-conjugated was used at 1:20 000, 1% non-fat Milk in TBS for 2h at RT. Washes were made as following:  after blocking, the membrane was washed 2X 5 min with 1x TTBS; after the primary antibody incubation, the membrane was washed 5x 5 min with 1x TTBS;  after the secondary antibody incubation, the membrane was washed 5x 5 min with 1x TTBS (contained Tween-20 at 500 µl/liter), followed by 3x 5 min with 1x TBS Wash Buffers (TBS and TTBS): 1X TBS (1 liter) 2.42 g Tris 8 g NaCl Adjust pH to 7.5-7.6 with HCl 1X TTBS: 1X TBS + 500 ul Tween-20 per liter. Reaction was developed following manufacture's recommendations and recorded using BioRad GelDoc.

Courtesy of Drs. Leonor Margalha/Elena Baena-González, Instituto Gulbenkian De Ciencia, Portugal

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 28940407

Journal: FEBS Lett

Figure Number: 1A,B

Published Date: 2017-11-01

First Author: Wurzinger, B., Mair, A., et al.

Impact Factor: 3.521

Open Publication

Arabidopsis AKIN10 but not CPK3 kinase activity is redox?sensitive. (A) In vitro phosphorylation of the 14?3?3 protein?binding site on NIA2 by AKIN10 and CPK3. Kinase reactions were done in the presence of 1 mm DTT or without DTT. Phosphorylation of NIA2 was visualised by western blotting with an antibody recognising a phosphorylated 14?3?3 protein?binding motive only. The coomassie brilliant blue (CBB)?stained membrane is shown below. Positions of the kinases and the substrate are indicated on the right?hand side by black arrowheads and bars. (B) In vitro phosphorylation of bZIP63 by AKIN10. GST?bZIP63 (bZIP63) was incubated with GST?AKIN10 (AKIN10) in kinase buffer containing 32P ?ATP and different concentrations of either DTT or H2O2. The proteins were separated by SDS/PAGE and phosphorylated proteins were detected by subsequent autoradiography. The CBB?stained gel is shown below. The positions of the full?length proteins are indicated. Relative quantification of the signals is shown in Fig. S1B.


Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 28940407

Journal: FEBS Lett

Figure Number: 3B,C

Published Date: 2017-11-01

First Author: Wurzinger, B., Mair, A., et al.

Impact Factor: 3.521

Open Publication

AKIN10 T?loop phosphorylation by SnAK2 is redox?dependent. (A) 3D?structural model of the AtSnRK1 complex based on the crystal structure of the rat RnAMPK ?1?1?1 heterotrimeric complex. The left image shows a superposition of the RnAMPK ?1?1?1 crystal structure and the derived AtSnRK1 model. RnAMPK ?1?1?1 is depicted in gold, AtAKIN10 in blue, AtAKIN?1 in red and AtSNF4 in pale?green. The T?loop (yellow) with T198 (green) and C200 (cyan) and the active site (magenta) are highlighted. The image on the right?hand side details the T?loop and active site part of SnRK1 in a sphere?representation with the spheres drawn to represent 1Ũ van der Waals radius (vdwr). (B) In vitro phosphorylation of AKIN10 by SnAK2. Inactive versions of GST?AKIN10 (K48M = AKIN10K/M and T198A = AKIN10T/A) were incubated with its upstream kinase GST?SnAK2 (SnAK2) in kinase buffer containing 32P ?ATP and different concentrations of either DTT or H2O2. The proteins were separated by SDS/PAGE and phosphorylated proteins were detected by subsequent autoradiography. The coomassie brilliant blue (CBB)?stained gel is shown below. The positions of the full?length proteins are indicated by arrowheads. Degradation products of GST?AKIN10 are marked by asterisks. (C) In vitro phosphorylation of the AKIN10 T?loop Threonine (T198) by SnAK2. Wild?type GST?AKIN10 (AKIN10 wt) or the C200S variant (AKIN10 C200S) was incubated with its upstream kinase GST?SnAK2 (SnAK2) in kinase buffer containing different concentrations of either DTT or H2O2. Threonine 198 phosphorylation in the T?loop of AKIN10 was visualised by western blotting with a phospho?specific antibody (??P?AMPK, top). The CBB?stained membrane is shown below.


Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 28940407

Journal: FEBS Lett

Figure Number: 4A,B

Published Date: 2017-11-01

First Author: Wurzinger, B., Mair, A., et al.

Impact Factor: 3.521

Open Publication

Intrinsic redox sensitivity of AKIN10 is retained in its T?loop phosphorylated state. (A,B) In vitro kinase activity (A) and phosphorylation (B) of AKIN10 under a simulated H2O2 burst. GST?AKIN10 (AKIN10 or inactive AKIN10K/M = K48M) was first mixed with either its substrate GST?bZIP63 (bZIP63) or its upstream kinase GST?SnAK2 (SnAK2) in kinase buffer containing 3.5 mm GSH. The kinase reactions were then started by adding 32P ?ATP and rising concentrations of H2O2 as indicated. The proteins were separated by SDS/PAGE and phosphorylated proteins were detected via autoradiography. The coomassie brilliant blue (CBB)?stained gel is depicted below. The positions of the full?length proteins are indicated by arrowheads. Degradation products of GST?AKIN10 are marked by asterisks. (C) Redox dependency of in vitro AKIN10 activity before and after phosphorylation by SnAK2. GST?AKIN10 (AKIN10) was first prephosphorylated (left panel) or not (right panel) by SnAK2 in kinase buffer containing 3.5 mm GSH. Then, GST?bZIP63 (bZIP63) and 32P ?ATP were added, either in the continued presence of 3.5 mm GSH (left lane) or in the presence of 3.5 mm GSH + 3.5 mm H2O2 (right lane). bZIP63 phosphorylation was analysed by autoradiography. The CBB?stained gel is depicted below. As activated AKIN10 is ~ 25 times more active than nonactivated AKIN10, the autoradiographs were developed separately in order to avoid oversaturation of the image from activated AKIN10.

Additional information

AKIN10 antibody works best when first the SnRK1 complex is immunorecipitated with this or another antibody and then anti-AKIN10 antibody is used to probe with, When this antibody is used against a lysate, the dominant rubisco protein can mask any immunoreactivity

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collection of antibodies to proteins involved in signal transduction

Plant protein extraction buffer

Background

Background

AKIN10 (E.C.= 2.7.11.1) is a catalytic subunit of the putative trimeric SNF1-related protein kinase (SnRK) complex, which may play a role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants. Synonymes: AKIN alpha-2

Product citations

Selected references Gutierrez-Beltran et al. (2021) Tudor staphylococcal nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis. EMBO J. 2021 Jul 21:e105043. doi: 10.15252/embj.2020105043. Epub ahead of print. PMID: 34287990.
Sun et al. (2021). Kinase SnRK1.1 Regulates nitrate channel SLAH3 Engaged in Nitrate-Dependent Alleviation of Ammonium Toxicity. Plant Physiol. 2021 Feb 9:kiab057. doi: 10.1093/plphys/kiab057. Epub ahead of print. PMID: 33560419.
Belda-Palazón et al. (2020) A dual function of SnRK2 kinases in the regulation of SnRK1 and plant growth. Nat Plants. 2020 Nov;6(11):1345-1353. doi: 10.1038/s41477-020-00778-w. Epub 2020 Oct 19. PMID: 33077877.
Krasnoperova et al. (2019). Potential Involvement of KIN10 and KIN11 Catalytic Subunits of the SnRK1 Protein Kinase Complexes in the Regulation of Arabidopsis ?-Tubulin. Cytology and Genetics, Volume 53, Issue 5, pp 349–356. (immunolocalization)
Blanco et al. (2019). Dual and dynamic intracellular localization of Arabidopsis thaliana SnRK1.1. J Exp Bot. 2019 Apr 15;70(8):2325-2338. doi: 10.1093/jxb/erz023.
Pedrotti et al. (2018). Snf1-RELATED KINASE1-Controlled C/S1-bZIP Signaling Activates Alternative Mitochondrial Metabolic Pathways to Ensure Plant Survival in Extended Darkness. Plant Cell. 2018 Feb;30(2):495-509. doi: 10.1105/tpc.17.00414. Epub 2018 Jan 18.
Frank et al. (2018). Circadian Entrainment in Arabidopsis by the Sugar-Responsive Transcription Factor bZIP63. Curr Biol. 2018 Aug 20;28(16):2597-2606.e6. doi: 10.1016/j.cub.2018.05.092.
Chan et al. (2017). SnRK1 phosphorylation of FUSCA3 positively regulates embryogenesis, seed yield, and plant growth at high temperature in Arabidopsis. Journal of Experimental Botany, erx233, https://doi.org/10.1093/jxb/erx233.
Crozet et al. (2016). SUMOylation represses SnRK1 signaling in Arabidopsis. Plant J. 2016 Jan;85(1):120-133. doi: 10.1111/tpj.13096.
Nukarinen et al. (2016). Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation. Sci Rep. 2016 Aug 22;6:31697. doi: 10.1038/srep31697.
Castro et al. (2015). SIZ1-Dependent Post-Translational Modification by SUMO Modulates Sugar Signalling and Metabolism in Arabidopsis thaliana. Plant Cell Physiol. 2015 Oct 14. pii: pcv149.
Emanuelle et al. (2015). SnRK1 from Arabidopsis thaliana is an atypical AMPK. Plant J. 2015 Mar 3. doi: 10.1111/tpj.12813.
Rodrigues et al. (2013). ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis. Plant Cell, Oct 31.

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