AKIN10 | SNF1-related protein kinase catalytic subunit alpha KIN10
AS10 919 clonality: polyclonal host: rabbit reactivity: Arabidopsis thaliana
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1 : 500 with standard ECL (WB)
|Expected | apparent MW||
|Predicted reactivity||Arabidopsis thaliana|
|Not reactive in||
Oryza sativa, Solanum lycopersicum, Vitis vinifera
|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.|
|Selected references||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.
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
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