Science Catalog 2012 Life Cell Signaling HisLink™ Spin Protein Purification System Product HisLink™ Spin Protein Purification System For Research Use Only. Not for Use in Diagnostic Procedures. Description: The HisLink™ Spin Protein Purification System provides a simple, quick and robust method of purifying polyhistidine- or HQ-tagged expressed proteins from E. coli using either a centrifuge- or vacuum-based method. Proteins can be purified directly from culture medium containing bacterial cells expressing polyhistidine- or HQ-tagged protein. The bacterial cells are lysed using the FastBreak™ Cell Lysis Reagent, followed immediately by the addition of the HisLink™ Protein Purification Resin to the culture. The addition of these reagents results in simultaneous bacterial lysis and binding of the polyhistidineor HQ-tagged proteins. Samples are then transferred to a Spin Column, where unbound protein is washed away and the target protein of interest is recovered by elution. Features: • Versatile: Choose between vacuum or centrifugation formats. • Convenient: Complete system containing all necessary components. • Productive: High binding capacity per column. Storage Conditions: Store the system at 4°C. Store Spin Columns, Collection Tubes and FastBreak™ Cell Lysis Reagent at room temperature. After reconstitution, store the DNase I in aliquots at –20°C. Protocol HisLink™ Spin Protein Purification System Technical Manual Biotin-Avidin Protein Purification SoftLink™ Soft Release Avidin Resin Product SoftLink™ Soft Release Avidin Resin For Research Use Only. Not for Use in Diagnostic Procedures. Description: SoftLink™ Avidin Resin can be used for the isolation and purification of biotinylated molecules. SoftLink™ Resin is a rigid, methacrylate polymeric gel filtration matrix, functionalized with covalently bound, monomeric avidin. Monomeric avidin binds biotin with a Kd value of 10–7M, allowing reversible binding of bound biotinylated proteins under mild elution conditions. Native, or tetrameric, avidin binds biotin with a very strong affinity (Kd = 10–15M), which in turn requires strong denaturing conditions for eluting bound material. Monomeric avidin allows the specificity of capture but also the mildness of release appropriate for the purification of sensitive biological materials. Features: • Sensitive: Binds 20–40nmol of biotinylated protein per milliliter of resin. • Easy to Use: Bound biotinylated molecules can be eluted under mild nondenaturing conditions (5mM biotin). • Versatile: Retains biotin binding ability following exposure to a wide range of pH, low or high ionic strength, 6M guanidine and 1% SDS. • Reusable: Regenerates at least 10 times without loss of binding capacity. • Robust: Supports high flow rates (300cm/hour) and centrifugal forces (1,500 × g) in batch applications. • Flexible: Purifications by batch or column method. Storage Conditions: Store at 4°C. Protocol SoftLink™ Soft Release Avidin Resin Product Information Part# 9PIV201 PinPoint™ Vector Sequencing Primer Product PinPoint™ Vector Sequencing Primer For Research Use Only. Not for Use in Diagnostic Procedures. Description: The PinPoint™ Vector Sequencing Primer is designed for sequencing inserts cloned into the PinPoint™ Xa Vectors (components of Cat.# V2020). The primer hybridizes upstream of the Factor Xa site at nucleotides 325–343, approximately 40–50 base pairs upstream of the multiple cloning region and can be used to determine if an insert is cloned in-frame with the biotinylation purification tag of the PinPoint™ Xa Vectors. The sequence of the PinPoint™ Vector Sequencing Primer is 5´-d(CGTGACGCGGTGCAGGGCG)-3´. It is supplied dried. Features: • Performance Tested: The PinPoint™ Vector Sequencing Primer is tested in double-stranded sequencing reactions with circular PinPoint™ Vectors. Storage Conditions: Store at –20°C. Size Cat.# Price (Fr) 2 µg V4211 135.00 Size Cat.# Price (Fr) 1 ml V2011 229.00 5 ml V2012 974.00 Part# TM281 Size Cat.# Price (Fr) 25 reactions V1320 324.00 PinPoint™ Xa Protein Purification System Product PinPoint™ Xa Protein Purification System Available Separately PinPoint™ Xa-1 Vector For Research Use Only. Not for Use in Diagnostic Procedures. Description: The PinPoint™ Xa Protein Purification System is designed for the production and purification of fusion proteins that are biotinylated in vivo. The DNA coding for the protein of interest is cloned into a PinPoint™ Vector downstream of a sequence encoding a peptide that becomes biotinylated in vivo. Biotinylated fusion proteins are produced in E. coli and are affinity-purified using the SoftLink™ Soft Release Avidin Resin. This proprietary resin allows elution of the fusion protein under nondenaturing conditions. The PinPoint™ Vectors feature the encoded endoproteinase Factor Xa (pronounced “ten a”) proteolytic site that provides a way to separate the purification tag from the native protein, and the vectors carry a convenient multiple cloning region for ease in construction of fusion proteins. The system contains vectors in all possible sense reading frames, an avidinconjugated resin, Streptavidin-Alkaline Phosphatase, a purification column and biotin. The PinPoint™ Xa Control Vector contains the chloramphenicol acetyltransferase (CAT) gene and is provided as a means of monitoring protein expression, purification and processing conditions. The system generally yields 1–5mg of protein per liter of culture. Features: • In vivo Biotinylation Tag: Allows purification of fusion proteins; many proteins produced have been soluble. • Easy to Use: Purification of biotinylated proteins with the SoftLink™ Resin can be performed by column or batch purification. • Easy Detection: Streptavidin Alkaline Phosphatase can be used to detect the biotinylated fusion protein in a pseudo-Western format to monitor purification. • Flexible: PinPoint™ Vectors are supplied for all reading frames. • Gentle Release Conditions: SoftLink™ Resin allows release of the fusion protein under nondenaturing conditions. • tac Promoter: Allows tightly regulated expression. Storage Conditions: Store the PinPoint™ Purification Column at room temperature. Store all remaining components at 4°C. The vectors may be stored at –20°C. Protocol PinPoint™ Xa Protein Purification System Technical Manual Part# TM028 Size Cat.# Price (Fr) 1 system V2020 1296.00 10 µg V2031 381.00 254 For complete and up-to-date product information visit: www.promega.com/catalog Pagina 257
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