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Kh232po4

Manufactured by PerkinElmer
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The KH232PO4 is a laboratory equipment product manufactured by PerkinElmer. It is designed to perform a specific function within the laboratory setting. The description of the core function of this product is limited to factual and unbiased information without any interpretation or extrapolation.

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Lab products found in correlation

Bas 2500, by GE Healthcare (2 mentions) Imaging plate, by Fujifilm (2 mentions) Optiphase hisafe 3, by PerkinElmer (1 mentions) Silica gel 60 g, by Merck Group (1 mentions) Tri carb 2100, by Hewlett-Packard (1 mentions) Kh2po4, by Merck Group (1 mentions) Typhoon fla 7000, by GE Healthcare (1 mentions)

5 protocols using kh232po4

1

Phosphate Uptake Quantification Protocol

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Cells seeded in four-well plates and transfected as described above were incubated for 48 h after the transfection. The cells were washed in 37 °C pre-warmed Pi-free DMEM medium (Gibco) and thereafter incubated at room temperature for 5 min in 37 °C pre-warmed Pi-free DMEM supplemented with KH232PO4 (NEX060, 1 Ci/mmol, Perkin Elmer) and cold Pi added to a final concentration of 1 mM Pi. They were then washed in 3× ice-cold 0.9% NaCl and lysed with 0.5% Triton X-100. One third of each lysate was transferred to counting vials, mixed with 5 mL liquid scintillation cocktail (Optiphase Hisafe 3 (Perkin Elmer)), and counted in a liquid scintillation counter. Dilution series of 32Pi were counted simultaneously. The remaining lysates were stored at −20 °C for later protein determination using the BCA™ Protein Assay Kit (Pierce).
Larsen F.T., Jensen N., Autzen J.K., Kongsfelt I.B, & Pedersen L. (2016). Primary Brain Calcification Causal PiT2 Transport-Knockout Variants can Exert Dominant Negative Effects on Wild-Type PiT2 Transport Function in Mammalian Cells. Journal of Molecular Neuroscience, 61(2), 215-220.
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2

Radiolabeling and TLC Analysis of Lipids

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Logarithmically growing cells were resuspended in 5 ml SC-Ura medium at the cell density (OD600 of 5) with 30 μCi KH232PO4 (PerkinElmer). After shaking incubation for 8 h at room temperature, lipids were extracted from cells by the method of Bligh and Dyer (1959) (link). Lipids spotted on a TLC plate (Silica gel 60G, Merck) were developed with chloroform/methanol/acetic acid (65:25:8, v/v/v) (Haselier et al., 2010 (link)) along with PG and CL (Sigma–Aldrich) as standards and radioactive spots were visualized by Imaging Plate (Fuji Film) and BAS-2500 (GE Healthcare). Unlabeled PG and CL were stained with 0.01% primuline in 80% (v/v) acetone and detected under UV light.
Hung C.H., Kobayashi K., Wada H, & Nakamura Y. (2016). Functional Specificity of Cardiolipin Synthase Revealed by the Identification of a Cardiolipin Synthase CrCLS1 in Chlamydomonas reinhardtii. Frontiers in Microbiology, 6, 1542.
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3

Radiolabeled Phosphate Uptake Assay

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Seedlings (7-d-old) of the wild-type, the Oslpr5 mutant generated using CRIPSR-Cas9, and the overexpression lines Ox3 and Ox6 were grown hydroponically under +P conditions for 7 d. The seedlings were then grown for different time periods (3–24 h for the mutant and 1–7.5 h for Ox3 and Ox6) in 200 ml of +P solution labeled with 32Pi (8 μCi of KH232PO4; Perkin-Elmer). The apoplastic 32Pi was then removed by incubating the roots of the seedlings in ice-cold desorption solution (2 mM MES, 0.5 mM CaCl2, and 0.1 mM NaH2PO4, pH 5.5) for 10 min. The seedlings were then blotted dry, and the roots and shoots were harvested separately, with their fresh weights being determined. The tissues were digested in a mixture of HClO4 and 30% (v/v) H2O2 at 28 °C for 8–12 h. A scintillation cocktail (3 ml) was added to the digested tissue and 32Pi activity was determined by using a liquid scintillation counter (Tri-Carb 2100, Packard). The 32Pi counts in the roots and shoots were used to determine the uptake rates and the shoot/root distribution ratio.
Ai H., Cao Y., Jain A., Wang X., Hu Z., Zhao G., Hu S., Shen X., Yan Y., Liu X., Sun Y., Lan X., Xu G, & Sun S. (2020). The ferroxidase LPR5 functions in the maintenance of phosphate homeostasis and is required for normal growth and development of rice. Journal of Experimental Botany, 71(16), 4828-4842.
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4

Anaerobic Phosphate Metabolism Assay

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Cells were cultured anaerobically in low phosphate glucose minimal
medium [0.5% glucose, 1.5 mM KH2PO4(pH 7.2), 2X MOPS (pH 7.2) 15 mM NaCl, 8.5 mM
(NH4)2SO4, 4 mM L-Cysteine, 5 ng
mL−1 haemin chloride, 100 mM MgCl2, 1.4 mM
FeSO4, 50 mM CaCl2, 1 mg mL−1vitamin K3, and 5 ng mL−1 vitamin
B12]. After reaching OD600=0.25,
960 μL of culture were removed from the anaerobic chamber for the
remainder of the experiment in a microcentrifuge tube, supplemented with 40
μL of 2550 μCi mL−1KH232PO4 (PerkinElmer, Waltham, MA,
USA), and incubated at 37°C for 2 hours. At this point, a 160
μl aliquot was removed and nucleotides were extracted. The remaining
culture was pelleted, resuspended in pre-warmed low phosphate minimal medium
without glucose, incubated for 1 hour at 37°C, and nucleotides were
extracted from 160 μl of culture. Nucleotides were extracted by the
addition of 40 μL 10 M ice-cold formic acid plus glass beads, 15
seconds vortexing, incubation on ice for 20 minutes, 5 seconds vortexing,
and incubation on dry ice for 20 minutes. These samples were thawed,
vortexed for 5 seconds, and centrifuged for 1 minute at 20,000 G to remove
cell debris. To analyze labeled nucleotides by thin-layer chromatography, 3
μL of extract was spotted on PEI-cellulose TLC sheets (Sigma) and
developed in 1.5 M KH2PO4 (pH 3.9). TLC plates were
imaged with a Typhoon Phosphoscanner and analyzed with QuantityOne
software.
Schofield W.B., Zimmermann-Kogadeeva M., Zimmermann M., Barry N.A, & Goodman A.L. (2018). The Stringent Response Determines the Ability of a Commensal Bacterium to Survive Starvation and to Persist in the Gut. Cell host & microbe, 24(1), 120-132.e6.
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5

Quantifying Phosphatidic Acid Levels

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To determine the PA level, freshly harvested floral buds of the WT and Pro35S:amiLPPα2-1 lppε1-2 lines #5 and #7 were immersed in ½ MS medium containing 30 μCi KH 2 32 PO 4 (PerkinElmer, Waltham, MA, USA). After incubating for 3 h under light, total lipids were extracted as described (Bligh and Dyer 1959; (link)Lin et al. 2019) (link), spotted on a thin layer chromatography plate and developed with the solvent system of chloroform:methanol:aqueous ammonia (120:80:8 by vol) for the first dimension and chloroform:methanol:acetic acid:water (170:20:15:3 by vol) for the second dimension. Radioactive spots were visualized using Imaging Plate (Fuji Film, Tokyo) with BAS-2500 (GE Healthcare, Chicago, IL, USA). The intensities were quantified with an image analyzer (Typhoon FLA 7000; GE Healthcare Life Sciences) and analyzed with Image J. Data are mean ± SD from three biologically independent experiments.
Nguyen V.C, & Nakamura Y. (2023). Distinctly localized lipid phosphate phosphatases mediate endoplasmic reticulum glycerolipid metabolism in Arabidopsis. The Plant cell, 35(5).
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