Lambda Protein Phosphatase (λ Protein Phosphatase) manufactured by our company is a Mn²⁺-dependent recombinant purified protein phosphatase. It catalyzes dephosphorylation of phosphorylated serine, threonine, tyrosine and histidine residues. This product is a 6×His-tagged recombinant λ phosphatase recombinantly expressed in Escherichia coli. The enzyme is encoded by open reading frame ORF221 of bacteriophage λ, and the encoded protein consists of 221 amino acid residues. Lambda Protein Phosphatase is applied for protein dephosphorylation, enabling research on the correlation between protein phosphorylation and protein activity/structure, as well as validation of the specificity of phospho-site antibodies.
| Source | Recombinantly expressed in E. coli
| | Molecular Weight | 26kDa | | Appearance | Sterile liquid
| | Storage Buffer | 50mM This-HCl , 100mM NaCl, 0.1mM MnCl₂, 0.1mM EGTA, 2mM Dithiothreitol (DTT), 0.01% Brij35, 50%(v/v) Glycerol, pH7.5 | | Enzyme Concentration | 70U/μL | | Purity | ≥95% | | Activity Definition | One unit (U) is defined as the quantity of enzyme that hydrolyzes 1 nmol of p-nitrophenyl phosphate within 1 minute at 30 °C and pH 7.5. |
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Components & Specifications
| L747583 | Component | 20KU | 5*20KU | Storage | | L747583A | Lambda Protein Phosphatase (70U/µl) | 300μl | 5*300μl | -20℃. Avoid freeze/thaw cycle. | | L747583B | 10× Lambda Protein Phosphatase Buffer | 1ml | 5*1ml | -20℃. Avoid freeze/thaw cycle. | | L747583C | 10× MnCl₂ (10mM MnCl₂) | 1ml | 5*1ml | -20℃. Avoid freeze/thaw cycle. |
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Product Applications:
Catalyze dephosphorylation of phosphorylated serine, threonine, tyrosine and histidine residues on target proteins.
Product Advantages:
Lambda Protein Phosphatase mediates protein dephosphorylation, which facilitates research on the correlation between protein phosphorylation and protein activity/structure. It can also be applied to verify the specificity of phospho-site-specific antibodies.
Instructions for Use:
1. Lambda Protein Phosphatase exhibits variable catalytic efficiency against different substrate proteins. Optimization of enzyme dosage and incubation time is recommended for individual experiments. The reaction system must contain 1× Lambda Protein Phosphatase Buffer supplemented with 1× MnCl₂ (final concentration: 1 mM).
2. Standard dephosphorylation reference system: In a total reaction volume of 50 μL, 100 U of Lambda Protein Phosphatase can remove approximately 100% phosphate groups from 5 μM monophosphorylated protein within 30 minutes.
3. Prepare the reaction system on ice according to the table below (50 μL reaction system as an example):
Reagent
| Volume
|
10× Lambda Protein Phosphatase Buffer
| 5μl
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| 10× MnCl₂ (10mM MnCl₂) | 5μl
|
| Protein Sample | ~μl
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| Lambda Protein Phosphatase (70U/µl) | 1μl (Optimization is recommended as needed)
|
| Ultrapure Water | Up to 50μl
|
| Total Volume | 50μl |
4. Mix thoroughly by pipetting, then perform a brief low-speed spin to collect the liquid at the bottom of the tube.
5. Incubation condition: incubate at 30 °C for 30 min.
6. Stop the reaction: add protein loading buffer and boil the sample, or process the sample according to the requirements of downstream experiments.
Precautions:
1. Repeated freeze-thaw cycles will reduce the activity of Lambda Protein Phosphatase. If long-term storage at −80°C is required, aliquot the enzyme into smaller portions in advance for convenient subsequent use. The reagent remains liquid at −20 °C but will freeze at −80°C.
2. 10 mM vanadate ions inhibit approximately 90% of Lambda Protein Phosphatase activity, and 50 mM EDTA inhibits roughly 95% enzyme activity. The following reagents do not interfere with enzyme activity: 1% Triton X-100, 0.4% Nonidet P-40, 0.025% Tween 20, 0.5 M NaCl, 0.1 mM ATP, 10 μg/mL pepstatin A, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 0.5 mM PMSF, 1 mM benzamidine.
3. The dephosphorylation rate varies greatly among different substrate proteins. The optimal reaction temperature for this enzyme is 30°C. When treating crude lysate samples with this enzyme, minimize incubation time and add an appropriate cocktail of protease inhibitors to reduce degradation of target proteins.
4. This product is for scientific research use by trained professionals only. It must not be used for clinical diagnosis or therapy, food or pharmaceutical manufacturing, nor stored in residential premises.
5. Wear a lab coat and disposable nitrile/latex gloves throughout all operations for personal safety and health.
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