PKA (Protein Kinase A) Activity Assay kit

Catalog No. ABIN2815091

Product Details

Target: PKA (Protein Kinase A (PKA))

Application: Kinase Activity Assay (KAA)

Purpose: The DetectX® PKA (Protein Kinase A) Activity kit is designed to quantitatively measure PKA ac- tivity in a variety of samples.

Brand: DetectX®

Sample Type: Cell Lysate, Tissue Samples

Cross-Reactivity (Details): PKAc alpha 100 %, PKAc beta 73 %, PKAc gamma 10.1 %,

Components: PKA Substrate 96 Well Plate 1 Plate Break-apart strip microtiter plate coated with PKA Substrate
PKA Standard 2 vials 5,000 Units of recombinant fully active PKA in special stabilizing buffer. One unit is defined as the amount of PKA required to catalyze the transfer of 1 pmol of ATP phosphate to substrate in 1 minute at 30°C. PKA Standard must be stored at -20°C. Read the instructions on the kit box and bottle about the temperature sensitive sensors.
ATP 1 vial ATP lyophilized. Store in supplied desiccator.
Phospho PKA Substrate Antibody 3 mL A solution of rabbit antibody specific for phospho-Substrate.
Goat anti-Rabbit IgG HRP Conjugate 3 mL A solution of goat antibody specific for rabbit IgG labeled with peroxidase.
Kinase Reaction Buffer Concentrate 60 mL A 2X concentrate containing detergents and stabilizers.
Cell Lysis Buffer 100 mL A Tris based buffer containing detergents. Store Frozen as this buffer contains no preservatives Cell Lysis Buffer must be stored at -20°C.
Wash Buffer Concentrate 30 mL A 20X concentrate that should be diluted with deionized or distilled water.
TMB Substrate 11 mL
Stop Solution 5 mL 1M solution of hydrochloric acid. CAUSTIC.
Plate Sealer 2 Each

Material not included: Distilled or deionized water.
Glass test tubes.
Shaking plate incubator capable of maintaining 30 °C.
Repeater pipet and disposable tips capable of dispensing 10, 25, 50 and 100 μL accurately.
The following Protease inhibitors MUST be added to all buffers that are used to measure PKA activity.
See pages 7 & 8. • Phenylmethanesulfonyl fluoride (PMSF), such as Sigma 78830 at 100 mM in ethanol. • A universal protease inhibitor cocktail (PIC) such as Sigma P1860 or Roche 05892970001.
In addition: • A phosphatase inhibitor, such as Sodium Orthovanadate (See activation instruction opposite.), or a phosphatase inhibitor cocktail, such as Sigma P5726, must added to the Cell Lysis buffer.
Colorimetric 96 well microplate reader capable of reading optical density at 450 and 650 nm.
Software for converting raw relative optical density readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting. DualRead™ System This kit uses our unique DualRead™ system.
We include instructions for an alternative high stan- dard which would typically generate ODs at 450 nm too high to be read on most plate readers.
By reading the plate at 650 nm (where TMB optical density is about 3 fold lower) immediately before addition of the Stop Solution some samples outside the normal standard curve range can be read.
See instructions on pages 8-10.

Application Details

Application Notes: This assay has been validated for Jurkat cell lysates.
Samples containing visible particulate should be centrifuged prior to using.

Protocol: A recombinant PKA standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
For samples that may have high PKA activity we offer an alternative protocol that reads the substrate reaction at 650 nm.
The kit utilizes an immobilized PKA substrate bound to a microtiter plate.
Samples containing PKA will, in the presence of the supplied ATP, phosphorylate the immobilized PKA substrate.
A rabbit antibody specific for the phospho-PKA substrate binds to the modified immobilized substrate.
An antibody specific for rabbit IgG labeled with peroxidase is then added to the plate to bind to the rabbit anti-phospho- PKA substrate.
After a short incubation and wash, substrate is added and the intensity of the color developed is directly proportional to the amount of PKA in the samples and standards.

Reagent Preparation:

Allow the kit reagents to come to room temperature for 30 minutes, except for the standard which must be kept on ice.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine PKA activity.
Keep all samples on ice and ensure they have been diluted appropriately prior to running them in the kit. kinaSe aSSay buffer - CRITICAL STEP! Dilute Kinase Reaction Buffer Concentrate 1:2 by adding one part of the concentrate to one part of deionized water.
Add 0.5 μL/mL of PIC and PMSF to 1 mM to make kinaSe aSSay buffer.
Use within 8 hours.
ATP Prior to opening vial, tap vial on bench top to ensure contents are in base of vial.
Add 1.2 mL of prepared kinaSe aSSay buffer to the ATP vial.
Vortex to solubilize.
Once diluted, store any unused ATP solution at -20 °C for up to 3 months.
Wash Buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water.
Once diluted this is stable at room temperature for 3 months.
Standard Preparation Spin down the contents of the PKA Standard vial in a microcentrifuge for 2 minutes at 14,000 rpm at 4 °C.
Keep all standards on ice during use.
Prepare an Intermediate Stock dilution by pipetting 1 mL of prepared kinaSe aSSay buffer into the PKA standard vial.
Invert vial and vortex thoroughly to ensure complete mixing of contents.
This Intermediate Stock will have an activity of 5,000 Units/mL.
Intermediate Stock is single use only.
Discard vial after preparing standards.
Label five tubes as #1 through #5.
Pipet the standards using the Intermediate Stock according to the table below.
The activity of PKA in tubes 1 through 5 will be 25, 20, 15, 10, and 5 Units/mL.
Alternative High Standard For samples that may exceed 25 U/mL prepare the 40 U/mL Alterna- tive Standard by pipetting 8 μL of Intermediate Stock into 992 μL of prepared kinaSe aSSay buffer.
Keep all Standards and Intermediate Stock on Ice and use within 30 minutes of preparation.
Standard Activity, U/mL Std 1 Std 2 Std 3 Std 4 Std 5 (25 U/ mL) (20 U/ mL) (15 U/ mL) (10 U/ mL) (5 U/ mL) kinaSe aSSay buffer (μL) 995 60 60 60 60 Addition 5 μL Intermediate 240 μL 180 μL 120 μL 60 μL Stock Std 1 Std 2 Std 3 Std 4

Sample Preparation:

Cells must be lysed in the Activated Cell Lysis Buffer, after addition of protease inhibitors and either activated orthovanadate or a phosphatase inhibitor cocktail to the provided Cell Lysis Buffer (see below). All cells and the lysates made from them must be stored at ≤ -70 °C and should be stored as aliquots for single use. Do not freeze-thaw samples. Do not store cells or lysates above -70 °C. The preparation of Activated Sodium Orthovanadate is as follows: Preparation of Activated Orthovanadate 200 mM Activated Orthovanadate should be prepared by dissolving 1.84 g of sodium or- thovanadate in 45 mL of water. Adjust the pH of the solution to 10 with 1M NaOH or HCl. At pH 10 the solution should be yellow. Boil the solution until it turns colorless (approxi- mately 10 min). All of the orthovanadate should dissolve. Cool to room temperature and readjust the pH to 10. Repeat the boiling of the solution and pH readjustment until the solution is colorless and remains at pH 10. Adjust the final volume to 50 mL with water. Store the Activated Sodium Orthovanadate in aliquots and freeze at -20 °C. Use an aliquot for preparing Activated Cell Lysis Buffer and discard. Preparation of Activated Cell Lysis Buffer Prepare the Activated Cell Lysis Buffer by addition of 1 μL of PIC per mL of Cell Lysate Buffer. Add 1 mM PMSF and 10 mM Activated Orthovanadate. The resulting Activated Cell Lysis Buffer is a pH 8 Tris based buffer containing 1 % NP-40 as a cell disruption agent. This assay may not be compatible with other cell lysis buffers containing high concentrations of SDS or other detergents and erroneous activity measurements may result. Cell Lysis PKA Activity: Jurkat Cell Lysate Dilution 18 Add prepared Activated Cell Lysis Buffer to the cells (for Jurkat cells, we lysed at 100 million cells per mL). 16 Incubate for 30 minutes on ice with occasional vor- 14 texing. Centrifuge at 10,000 rpm at 4 °C for 10 min- 12 utes and carefully aspirate off the supernatant for 10 analysis. Supernatants can be frozen at ≤ -70 °C for later analysis. 8 6 The supernatants should be diluted at least 1:10 into Obs. PKA Activity (U/mL)4 prepared kinaSe aSSay buffer (see Page 8) prior to 2 running in the assay. It is recommended that a con- trol lysate be serially diluted in kinaSe aSSay buffer to 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 determine the appropriate dilution to obtain a linear Jurkat Cell Lysate Diln (x 10^6)/mL response. See Graph at right. Samples diluted in kinaSe aSSay buffer can be frozen at ≤ -70 °C for analysis later.

Assay Procedure:

Determine the number of wells to be used and return unused wells to the foil ziploc bag with the desiccant. Seal and store at 4 °C.
1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification.
2. Pipet 40 μL of kinaSe aSSay buffer as a Zero Standard into duplicate wells in the plate.
3. Pipet 40 μL of samples or standards diluted in kinaSe aSSay buffer into duplicate wells in the plate.
4. Add 10 μL of the reconstituted ATP to each of the wells using a repeater pipet.
5. Seal the plate and incubate at 30 °C shaking for 90 minutes.
6. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
7. Add 25 μL of the Goat anti-rabbit IgG HRP conjugate to each well using a repeater pipet.
8. Add 25 μL of the Rabbit Phospho PKA Substrate antibody to each well using a repeater pipet.
9. Seal the plate and incubate the plate at room temperature for 60 minutes with shaking. 10. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels. 11. Add 100 μL of the TMB Substrate Solution to each well, using a repeater pipet. 12. Incubate the plate at room temperature for 30 minutes. DualRead™ If the blue substrate color of any of your samples appears darker than the 25 U/mL standard we recommend reading the plate at 650 nm, immediately prior to adding stop solution. 13. Add 50 μL of the Stop Solution to each well, using a repeater pipet and read the optical density generated from each well in a plate reader capable of reading at 450 nm. 14. Use the plate reader's built-in 4PLC software capabilities to calculate PKA activity for each sample. NOTE: If you are using only part of a plate, at the end of the assay through away the used wells and retain the white plate frame for use with the remaining unused wells.

Calculation of Results:

Average the duplicate 450 nm (and optional 650 nm) OD readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean ODs for the zero standard.
The sample activity obtained should be multiplied by the dilution factor to obtain neat sample values. typical data (450 nm read) Sample Mean OD Net OD PKA Activity (450nm) (450nm) (U/mL) Standard 1 1.272 1.223 25 Standard 2 0.929 0.880 20 Standard 3 0.618 0.569 15 Standard 4 0.363 0.314 10 Standard 5 0.155 0.106 5 Zero 0.049 0.000 0 Sample 1 0.158 0.109 5.91 Sample 2 1.000 0.951 20.9 Or use the online tool from http://www.myassays.com/arbor-assays-protein-kinase-a-activity-kit. assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data (650 nm read) Sample Mean OD Net OD PKA Activity (650nm) (650nm) (U/mL) Alt.
Std. 0.564 0.528 40 Standard 1 0.42 0.384 25 Standard 2 0.307 0.271 20 Standard 3 0.23 0.195 15 Standard 4 0.102 0.066 10 Standard 5 0.06 0.025 5 Zero 0.036 0 0 Sample 1 0.274 0.238 18.8 Sample 2 0.109 0.073 7.86 ® Always run your own standard curve for calculation of results.
Do not use this data. www.ArborAssays.com 10 WEB INSERT 151105 Typical Standard Curves 450 nm Read 1.4 1.2 1.0 0.8 OD0 (.6450 nm) 0.4 0.2 0.0 0 5 10 15 20 25 PKA Activity (U/mL) 650 nm Read 0.6 0.5 0.4 0.3 Net OD (650nm) 0.2 0.1 0 0 5 10 15 20 25 30 35 40 PKA Activity (U/mL)

Restrictions: For Research Use only

Handling

Preservative: Sodium azide

Precaution of Use: The PKA Standard and Cell Lysis Buffer MUST be stored at -20°C.
As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The coated plate needs to be stored desiccated.
The silica gel pack included in the foil ziploc bag will keep the plate dry.
The silica gel pack will turn from blue to pink if the ziploc has not been closed properly.
This kit utilizes a peroxidase-based readout system.
Buffers, including other manufacturers Wash Buffers, containing sodium azide will inhibit color production from the enzyme.
Make sure all buffers used for samples are azide free.
Ensure that any plate washing system is rinsed well with deionized water prior to using the supplied Wash Buffer as prepared on Page 8.
The Stop Solution is acid.
The solution should not come in contact with skin or eyes.
Take appro- priate precautions when handling this reagent.

Storage: -20 °C,4 °C,RT

Storage Comment: The unopened kit should be stored at -20°C until the expiration date of the kit. Once opened the kit can be stored at 4°C up to the expiration date on the kit label, except for the PKA Standard and Cell Lysis Buffer which must be stored at -20°C. The Cell Lysis Buffer has no preservative and must be kept frozen at -20°C. All components of this kit can be stored together at -20°C. The kit must be used prior to the expiration date on the kit box label.

References

Chen, Zheng, Liu, Lin, Liao, Xin, Zhong, Cheng, Zhang: "Adiponectin Inhibits TNF-α-Activated PAI-1 Expression Via the cAMP-PKA-AMPK-NF-κB Axis in Human Umbilical Vein Endothelial Cells." in: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, Vol. 42, Issue 6, pp. 2342-2352, (2017) (PubMed).

Tetzner, Gebolys, Meinert, Klein, Uhlich, Trebicka, Villacañas, Walther: "G-Protein-Coupled Receptor MrgD Is a Receptor for Angiotensin-(1-7) Involving Adenylyl Cyclase, cAMP, and Phosphokinase A." in: Hypertension (Dallas, Tex. : 1979), Vol. 68, Issue 1, pp. 185-94, (2016) (PubMed).

Sinha, Ren, Arora, Moon, Yarlagadda, Woodrooffe, Lin, Schuetz, Ziady, Naren: "PKA and actin play critical roles as downstream effectors in MRP4-mediated regulation of fibroblast migration." in: Cellular signalling, Vol. 27, Issue 7, pp. 1345-55, (2015) (PubMed).

Baliño, Ledesma, Aragon: "Role of phosphodiesterase-4 on ethanol elicited locomotion and narcosis." in: Neuropharmacology, Vol. 101, pp. 271-8, (2015) (PubMed).

Ul Kabir, Samad, Ahmed, Jahan, Akhter, Tasnim, Hasan, Sayfe, Hannan et al.: "Aqueous fraction of beta vulgaris ameliorates hyperglycemia in diabetic mice due to enhanced glucose stimulated insulin secretion, mediated by acetylcholine and GLP-1, and elevated glucose uptake via ..." in: PLoS ONE, Vol. 10, Issue 2, pp. e0116546, (2015) (PubMed).

Yang, Liu, Tu, Wan, Zhang, Wu, Chen, Zhou, Mu, Wang: "Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF." in: Nature communications, Vol. 5, pp. 5627, (2014) (PubMed).

Purohit, Hu, Chen, Whelan, Moustaid-Moussa, Zhao: "Activation of nucleotide oligomerization domain containing protein 1 induces lipolysis through NF-κB and the lipolytic PKA activation in 3T3-L1 adipocytes." in: Biochemistry and cell biology = Biochimie et biologie cellulaire, Vol. 91, Issue 6, pp. 428-34, (2013) (PubMed).

Target Details

Target: PKA (Protein Kinase A (PKA))

Alternative Name: Protein Kinase A (PKA Products)

Synonyms: PKCD Kit, Pkaca Kit, Cs-PKA Kit, PKCA1 Kit, PKA Kit, protein kinase, cAMP dependent, catalytic, alpha Kit, protein kinase cAMP-activated catalytic subunit alpha Kit, Prkaca Kit, PRKACA Kit

Background: PKA was discovered in the laboratory of Edwin G. Krebs in the 1960's1. This important class of kinases, refered to as Arg-directed kinases or AGC-family kinases, includes cAMP-dependent protein kinase (PKA or cAPK), cGMP-dependent protein kinase (PKG), protein kinase C, Akt and RSK. These kinases share a substrate specificity characterized by Arg at position 3 relative to the phosphorylated serine or threonine2-4. The second messenger cyclic AMP (cAMP) activates PKA in mammalian cells and controls many cellular mechanisms such as gene transcription, ion transport, and protein phosphorylation2. Inactive PKA is a heterotetramer composed of a regulatory subunit (R) dimer and a catalytic subunit (C) dimer. In this inactive state, the pseudosubstrate sequences on the R subunits block the active sites on the C subunits. PKA shares substrate specificity with Akt (PKB) and PKC4. Substrates that present this consensus sequence and are phosphorylated by PKA are Bad (Ser155), CREB (Ser133), and GSK-3 (GSK-3α Ser21 and GSK-3ß Ser9) 5-7. PKA has been implicated in numerous cellular processes, including modulation of other protein kinases, regulation of intracellular calcium concentration, and regulation of transcription8. Tran- scriptional responses to increased cAMP occur through activation of the cAMP response element- binding protein (CREB), cAMP response element modulator (CREM), and activating transcription factor 1 (ATF1) 9. Each of these transcription factors contains a kinase-inducible domain contain- ing a conserved site for phosphorylation by PKA