免疫化学/PKA激酶分析试剂盒，I/96型分析/试剂盒/ICP0216
商品编号：
ICP0216
品牌：
immunechem
市场价：
¥9900.00
美元价：
4950.00
产品分类：
缓冲液
公司分类：
buffer_solution
联系Q Q：
3392242852
电话号码：
4000-520-616
电子邮箱：
info@ebiomall.com
商品介绍
PKA Kinase Assay Kits, Type I
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KITCOMPONENTS
1. PKA substrate plate: a 96 well plate with 12x8 removable strips. Each well was pre-immobilized with 50μL ofPKA-substrate (KRREILSRRPSYR).
2. Anti-pSub (pCreb) antibodies (rabbit, affinity purified): 1μg/mL, 5mL.
3. Kinase Assay Dilution buffer: 10mL.
4. Antibody dilution buffer: 10mL.
5. Goat anti-rabbit IgG HRP conjugates: Catalog# ICP9803, 0.5mg/mL in HRP stabilizing buffer, 30μL. Dilute to 0.5μg/mL (1:5000 dilution) with antibody dilution buffer (D) as working solution.
6. Adenosine tri-phosphate (ATP): 2mg. Reconstitute with 2mL kinase assay dilution buffer (C) as working solution.Once it is dissolved in the buffer, store at -20ºC for better stability.
7. Peroxidase substrate: 10mL, TMB (3,3"-5,5"-Tetramethylbenzidine).
8. Stop solution: 10mL.
9. 10x PBSt: 10mL.
KITDESCRIPTION
Quality control: The kits were tested using PKA from Sigma (#P5511). Sensitivity is approximately 100ng of PKA/assay.
Storage and Stability: Stable for 6 months at 4ºC from date of shipment. Avoid the light and heat.
Descriptions: PKA is a cyclic AMP dependent protein kinase. The PKA kinase assay kit (Type I) is a non-radioactive, homogenous, simple, rapid, antigen-captured immunosorbent assay. This assay is designedfor the assay of PKA in solution. The principle of the assay is simple. Purified or partially purified PKA will phosphorylate the PKA substrate on the 96-well plate with the presence of ATP. The phosphorylated substrate (pSubstrate) is then analyzed with the anti-phosphosubstrate antibodies. Subsequently, the binding anti-pSub antibodies will be quantified with the secondary antibody-HRP conjugate, which generates the color signal from its substrate, TMB (OD at 450 nm). The final color intensity is proportional to the initial PKA phosphorylation activity.
SUMMARYOFTHEASSAY
1. Kinase reaction: KRREILSRRPSYR + ATP + Kinase → KRREILSRRPpSYR
2. The phosphorylated substrate on the plate will be detected with an anti-pSubstrate antibody:KRREILSRRPSYR (no antibody binding), KRREILSRRPpSYR (antibody binds to the plate)
3. Signal Generation: Secondary antibody binds to antibody-KRREILSRRPpSYR complex formed on the plate.
PROTOCOL
1. Soak the well of the substrate plate (component A) with 50μL of kinase assay dilution buffer (component C) for 10 mins, then aspirate.
2. Prepare purified or partially purified PKA or PKA-inhibitor mixture in 30μL of the kinase assay dilution buffer to the well of the substrate plate as prepared in step 1 (Note: For preparation of the positive control, refer to the Appendix)
3. Add samples to the appropriate wells.
4. Add 10μL of the ATP (component F) solution to the wells to initiate the kinase reaction at 30-35ºC for 90 mins. Hand-shake the strip every 20 mins. If a shaker with rotate angle is applied, the speed arranged at 60 rounds per min should be adequate.
5. Empty the wells then add 40μL of the anti-pSubstrate antibodies (component B) at room temperature for 60 mins. Shake every 20 mins.
6. Empty the wells and fill the well with 100μL of PBSt washing buffer. Let the washing buffer stay for 1-2 mins then aspirate. Repeat this washing step four times.
7. Add 40μL of goat anti-rabbit IgG HRP (component E) working solution to the well and incubate at room temperature for 30 mins. Shake the wells every 10 mins.
8. Empty the wells and repeat the washing as step 6.
9. Add 60μL of TMB to develop the color (for 30-60 mins).
10. Stop the reaction with 20μL of the stop solution (2 N HCl).
11. Calculation of the relative kinase activity:OD (sample)-OD(negative)/ng of kinase
QUALITYCONTROLTEST
Assay for activity of PKA (Sigma#P5511, lot#108H7846)
Activity, transfer 1 picomole of phosphate to kemptide/min/μg of protein.
APPENDIX
Preparation of Positive Control
Sample Preparation of Recombinant Kinase
1. Label 5 microfuge tubes; 1-4, plus a negative control. Note: The negative control should only contain assay dilution buffer; no kinase.
2. Aliquot 38μL of assay dilution into tube#1 and 30μL into tubes 2-4.
3. Add 2μL of recombinant kinase into tube#1 and mix thoroughly.
4. Transfer 10μL from tube#1 to tube#2 and mix.
5. Transfer 10μL from tube#2 to tube#3 and mix.
6. Transfer 10μL from tube#3 to tube #4 and mix.
7. Transfer 30μL from each of the mixtures (tubes 1-4 plus the control) into the appropriate wells of the substrate plate.
Product Specific References |
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品牌介绍
加拿大免疫化学药剂有限公司(ImmuneChem Pharmaceuticals Inc. 简称ICP) 是由加拿大政府资助而创立的生物高科技开发公司。始创立于1997年，公司的研究与开发部门位于加拿大不列颠斯哥伦比亚省大温哥华地区。ICP是一个以蛋白质化学技术和免疫技术为基础的免疫化学药物高科技公司。以固相表面化学技术为基础，以蛋白质分离提纯基础为核心，以免疫技术为应用，致力于开发科研和诊断用免疫球蛋白以及医疗用被动免疫球蛋白，其所创建的ImmuneChem品牌抗体试剂多年来畅销世界各地。
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