GFP‐nAb™ Polyacrylamide resin is a durable, ready‐to‐use medium for immobilized affinity purification (gravity, FPLC, HPLC) featuring our single‐domain GFP antibody (GFP‐nAb™). Its very high affinity and specificity for GFP results in virtually background‐free purification. GFP‐nAb™ binds nearly all fusions to fluorescent proteins derived from Aequorea victoria under native conditions, along with bound interacting partners. Medium properties Matrix Spherical Polyacrylamide Average Particle Size 65 µm Binding Capacity 1mg EGFP/ml resin Max Linear Flow Rate 3000cm/hr Max Pressure 100 p.s.i. (6.9 bar) Chemical Stability All commonly used buffers, Ethanol ≤ 20%, NaCl ≤ 2M pH Stability Short Term: 2 – 12; Long Term: 4 ‐ 10 Storage PBS pH 7.4 with 20% Ethanol Storage Temperature 4OC GFP‐nAb™ Polyacrylamide is provided as a 50% slurry in storage buffer. For column packing and automated purification parameters, follow manufacturer's recommendations. General Considerations GFP‐nAb™ Polyacrylamide resin will strongly bind GFP‐tagged proteins under a wide variety of buffer conditions. Affinity for GFP is essentially constant from 0 – 2M NaCl and pH 4 to 10. Binding occurs rapidly, generally within the first few minutes of exposure to the resin; however, for very dilute samples we recommend longer incubation times (or lower flow rates) to ensure complete binding. All buffers and samples should be passed through a 0.22µm filter prior to being applied the resin to prevent clogging by particulate material. When changing buffer conditions, it is important to equilibrate the resin until absorbance stabilizes before applying the sample. Reducing agents such as ME, DTT, or TCEP may cause some loss of binding affinity and should be avoided in all buffers whenever possible. Low concentrations of ME (~1‐5 mM) may be used if absolutely necessary. High concentrations of detergents (especially ionic detergents such as SDS) may cause nonspecific binding to the matrix and should be avoided. During binding, detergent concentration should be < 0.5%. Washes should be performed with up to 2M NaCl if tolerated by your GFP fusion protein and its binding partners. When purifying complexes, it may be advisable to use lower salt concentrations for washing. GFP-nAb™
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Elution from GFP‐nAb™ Polyacrylamide is best accomplished using an acidic buffer, and reaches quantitative levels around pH 3. We recommend eluting with 0.1M sodium citrate pH 2.5. Eluate should be monitored for absorbance at 280nm or 400nm to ensure complete removal of GFP from the resin. Neutralization of the column prior to complete elution will result in re‐binding. Immediately following elution, re‐equilibrate the column in a neutral buffer. Prolonged exposure to pH < 4 may decrease the binding capacity of the resin by irreversibly denaturing the GFP‐nAb™ protein. See below for column regeneration parameters. Sample preparation Prepare cell or tissue lysate via your preferred method. We strongly recommend including protease inhibitors in the lysis buffer. Dilute the sample with binding buffer to ≥ 25 column volumes. Filter the sample through a 0.22µm membrane prior to applying it to the resin. Purification The following is an example protocol for FPLC‐based purification of a GFP fusion protein from cell lysate diluted in binding buffer to 25 column volumes. Parameters should be determined empirically for individual GFP fusion proteins and your preferred mode of purification. Follow manufacturer protocols for chromatography column packing and preparation. For high flow rates (> 500 cm/hr) the resin should be packed at a flow rate ~25% higher than the intended operational flow rate for a minimum of 10 column volumes. 1. Equilibrate resin by washing with ~40 column volumes of binding buffer at a flow rate of ~180 column volumes/hr (a linear velocity of ~150‐200 cm/hr for a 1cm diameter column). 2. Apply prepared sample to GFP‐nAb™ resin, decreasing the flow rate to ~60 column volumes/hr. Absorbance at 488nm or fluorescence (488nm excitation, ≥ 510 nm emission) in the flowthrough indicates that the GFP binding capacity of the column has been reached. 3. Wash with wash buffer until absorbance at 280nm reaches a stable value. 4. Elute with elution buffer, monitoring absorbance at 280nm or 400nm (specific to GFP). Some elution may occur as the pH value declines, with complete elution when entire column has reached pH 2.5 – 3. Elute until absorbance reaches a stable value, then immediately neutralize resin with neutralization buffer. Regenerating the resin When treated and stored as recommended, GFP‐nAb™ Polyacrylamide resin may be re‐used 20+ times without a substantial decrease in binding capacity. No GFP should remain bound to the resin after a properly performed elution. However, neutralization prior to complete dissociation may lead to residual GFP. Depending on the sample type being processed, protein nonspecifically bound to the matrix may also build up over time. GFP-nAb™
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If the column must be cleared, flush with 5 column volumes of elution buffer followed by 10 column volumes of neutralization buffer. Repeat this sequence once more, wash with 10 column volumes of 2M NaCl, then re‐equilibrate with a low‐salt, neutral pH buffer (such as the recommended binding buffer). This procedure will clear the resin of any remaining GFP and other nonspecifically bound proteins. Storage Store the resin at 4OC. If the column is to be used within 24 hours, it may be stored in a neutral binding buffer, preferably with constant slow flow (~0.1ml/min). For longer‐term storage, we recommend use of a preservative such as 0.05% sodium azide or 20% ethanol. Equilibrate the resin in 20 – 40 column volumes of storage buffer prior to capping the column. Suggested buffer compositions (1X) Binding Buffer 20mM Tris‐HCl, pH 7.5 150mM NaCl Wash Buffer 20mM Tris‐HCl, pH 7.5 0.5 – 2M NaCl Elution Buffer 0.1M sodium citrate, pH 2.5 Neutralization Buffer 50mM Tris‐HCl, pH 8 Storage Buffer PBS or 20mM Tris‐HCl, pH 7.2 – 7.5 20% ethanol GFP-nAb™
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