in vitro peptag pka activity assay (Promega)

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Promega in vitro peptag pka activity assay

Inhibition of AS Tpk1 and Tpk2 by 1NM-PP1 blocks C. albicans growth and hyphal morphogenesis. (A) A flowchart outlining the strain construction strategy used to make the TPK1 and TPK2 AS mutants. A black arrow indicates deletion or complementation at both alleles using a transient CRISPR strategy . Two orange arrows indicate the sequential complementation of both alleles using homologous recombination. The marker code utilizes gray to indicate auxotrophy for the indicated amino acid or sensitivity to the indicated drug. Red indicates prototrophy for the amino acid or resistance to the drug indicated. (B) Treatment with 1NM-PP1 inhibits the growth of AS TPK1 or TPK2 strains. C. albicans strains were grown overnight to saturation in YPD and then diluted to an optical density at 600 nm (OD 600 ) of 0.002 into fresh YPD containing either 2.5 μM 1NM-PP1 or dimethyl sulfoxide (DMSO) vehicle control. Strains were grown at 30°C for 24 h and growth was assessed by OD 600 (see color bar). (C) Treatment with 2.5 μM 1NM-PP1 inhibits filamentation of the AS TPK2 strain. Strains were grown at 37°C in either YPD with 10% serum; Spider medium; or yeast nitrogen base supplemented with 11 mM glucose, 2% casamino acid, and 5 mM GlcNAc (YNBNAG) for 4 h. (D) Treatment with 5 μM 1NM-PP1 inhibits filamentation of the AS TPK1 and TPK2 strains in RPMI with 10% serum and 5% CO 2 at 37°C. Strains were grown for 4 h. (E) TPK1 expression and TPK2 expression are not significantly different in wild-type or AS C. albicans strains. Transcript levels were normalized to ACT1 . Data are presented as mean ± SD of technical triplicates. (F) A strain of C. albicans with both AS TPK1 and TPK2 alleles exhibited analog-specific inhibition of growth. C. albicans strains were grown as described in B. (G) Treatment with 1NM-PP1 inhibits filamentation in a strain of C. albicans with both AS TPK1 and TPK2 alleles. Strains were grown in YNBNAG at 37°C for 4 h. (H) Silver staining of the purified Tpk2/Bcy1 holoenzyme. Strains were grown to log phase in YPD, and then the <t>PKA</t> holoenzyme was purified using a flag-affinity purification as described previously . HF represents His6-Flag3. The purified product was run on a 10% SDS-PAGE gel and silver stained. *, impurities. (I) PKA kinase assays were performed using the <t>Peptag</t> kit (Promega). The nonphosphorylated peptide contains one positive charge (+1) and migrates toward the cathode. The phosphorylated peptide contains one negative charge (−1) and migrates toward the anode. Products were resolved on a 0.8% agarose gel in 50 mM Tris-HCl (pH 8.0). The gel was imaged with an Alexa 568 channel. In all panels, the acronym AS stands for analog sensitive. All experiments were performed in biological duplicate.

In Vitro Peptag Pka Activity Assay, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pka+buffer/peptag+pka+reaction+buffer/pmc10286698-44-3-8
Average 90 stars, based on 1 article reviews

in vitro peptag pka activity assay - by Bioz Stars, 2026-11

90/100 stars

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1) Product Images from "Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1"

Article Title: Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1

Journal: mSphere

doi: 10.1128/msphere.00095-23

Figure Legend Snippet: Inhibition of AS Tpk1 and Tpk2 by 1NM-PP1 blocks C. albicans growth and hyphal morphogenesis. (A) A flowchart outlining the strain construction strategy used to make the TPK1 and TPK2 AS mutants. A black arrow indicates deletion or complementation at both alleles using a transient CRISPR strategy . Two orange arrows indicate the sequential complementation of both alleles using homologous recombination. The marker code utilizes gray to indicate auxotrophy for the indicated amino acid or sensitivity to the indicated drug. Red indicates prototrophy for the amino acid or resistance to the drug indicated. (B) Treatment with 1NM-PP1 inhibits the growth of AS TPK1 or TPK2 strains. C. albicans strains were grown overnight to saturation in YPD and then diluted to an optical density at 600 nm (OD 600 ) of 0.002 into fresh YPD containing either 2.5 μM 1NM-PP1 or dimethyl sulfoxide (DMSO) vehicle control. Strains were grown at 30°C for 24 h and growth was assessed by OD 600 (see color bar). (C) Treatment with 2.5 μM 1NM-PP1 inhibits filamentation of the AS TPK2 strain. Strains were grown at 37°C in either YPD with 10% serum; Spider medium; or yeast nitrogen base supplemented with 11 mM glucose, 2% casamino acid, and 5 mM GlcNAc (YNBNAG) for 4 h. (D) Treatment with 5 μM 1NM-PP1 inhibits filamentation of the AS TPK1 and TPK2 strains in RPMI with 10% serum and 5% CO 2 at 37°C. Strains were grown for 4 h. (E) TPK1 expression and TPK2 expression are not significantly different in wild-type or AS C. albicans strains. Transcript levels were normalized to ACT1 . Data are presented as mean ± SD of technical triplicates. (F) A strain of C. albicans with both AS TPK1 and TPK2 alleles exhibited analog-specific inhibition of growth. C. albicans strains were grown as described in B. (G) Treatment with 1NM-PP1 inhibits filamentation in a strain of C. albicans with both AS TPK1 and TPK2 alleles. Strains were grown in YNBNAG at 37°C for 4 h. (H) Silver staining of the purified Tpk2/Bcy1 holoenzyme. Strains were grown to log phase in YPD, and then the PKA holoenzyme was purified using a flag-affinity purification as described previously . HF represents His6-Flag3. The purified product was run on a 10% SDS-PAGE gel and silver stained. *, impurities. (I) PKA kinase assays were performed using the Peptag kit (Promega). The nonphosphorylated peptide contains one positive charge (+1) and migrates toward the cathode. The phosphorylated peptide contains one negative charge (−1) and migrates toward the anode. Products were resolved on a 0.8% agarose gel in 50 mM Tris-HCl (pH 8.0). The gel was imaged with an Alexa 568 channel. In all panels, the acronym AS stands for analog sensitive. All experiments were performed in biological duplicate.

Techniques Used: Inhibition, CRISPR, Homologous Recombination, Marker, Control, Expressing, Silver Staining, Purification, Affinity Purification, SDS Page, Staining, Agarose Gel Electrophoresis

Activity Assay:

Article Title: Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production
Article Snippet: .. Total protein was extracted as described previously and the Peptag cAMP dependent PKA activity assay (Promega) performed according to manufacturer’s instructions. .. Quantification of the intensity of the phosphorylated substrate was determined via densitometry analysis using the ImageJ software.

Article Title: The low affinity glucose transporter HxtB is also involved in glucose signalling and metabolism in Aspergillus nidulans
Article Snippet: .. Cellular protein extracts were prepared according to ref. . A Bradford Assay was carried out to quantify the protein concentration in the samples (Bio-Rad, according to manufacturer’s instructions). cAMP concentrations and PKA activities were measured in the cell extracts using the Amersham cAMP Biotrak EIA system assay (GE Healthcare) and the Peptag cAMP dependent PKA activity assay (Promega) according to manufacturer’s instructions. .. The phosphorylated substrate was then run on a 1% w/v agarose gel and the intensity of the band was quantified using ImageJ.

Article Title: Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1
Article Snippet: .. Using an in vitro PepTag PKA activity assay (Promega), we confirmed that the Tpk2/Bcy1 holoenzymes phosphorylate a fluorescent peptide substrate in the presence of cAMP. ..

other:

Article Title: G-protein coupled receptor-mediated nutrient sensing and developmental control in Aspergillus nidulans.
Article Snippet: The Amershan cAMP Biotrak EIA system assay (GE Healthcare) and the Peptag cAMP-dependent PKA activity assay (Promega) were performed according to manufacturer’s instructions.

Incubation:

Article Title: Alcohol Withdrawal Increases Protein Kinase A Activity in the Rat Inferior Colliculus.
Article Snippet: .. To start the reaction, an aliquot of the PKA sample was incubated for 30 minutes at room temperature in PepTag PKA reaction buffer containing 0.4 lg/ll Kemptide PepTag A1 (L-R-R-A-S-L-G; Promega). ..

Bradford Assay:

Protein Concentration:

Enzyme-linked Immunosorbent Assay:

In Vitro:

Article Title: Gli protein activity is controlled by multi-site phosphorylation in vertebrate Hedgehog signaling
Article Snippet: .. In vitro phosphorylation was carried out on immunoprecipitated proteins in the PKA reaction buffer (Promega) in the presence of 0.5mM ATP, 10μCi of [γ- 32 P]ATP, and 21 units of PKA (NEB) for 30 min at 30°C. ..

Phospho-proteomics:

Immunoprecipitation:

Kinase Assay:

Article Title: WAVE1 mediates suppression of phagocytosis by phospholipid-derived DAMPs
Article Snippet: .. PKA kinase assay (PepTag; Promega) was performed according to the manufacturer’s instructions. ..

Review