TITLE: Anti-c-Myc Tag (9E10) Affinity Gel Protocol
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Catalog Number: </span><span>658502</span></div><div class = "text-block"><span style = "font-weight:bold;">Storage Temperature: </span><span>2°C-8°C</span></div><div class = "text-block"><span style = "font-weight:bold;">Product Description: </span><span>Anti-c-Myc Tag (9E10) Affinity Gel consists of anti-c-myc monoclonal antibody (clone 9E10), covalently immobilized onto 6% high density glyoxal agarose beads. The affinity resin can be used in affinity purifying and immunoprecipitation of c-Myc-tagged fusion protein.</span></div><div class = "text-block"><span style = "font-weight:bold;">Binding Specificity: </span><span>Mouse monoclonal antibody 9E10 recognizes the c-myc epitope N-EQKLSEEDL-C and is purified through Protein G chromatography. The antibody conjugated affinity resin can bind to epitope at N-terminal, C-terminal, and internal locations of a fusion protein. The binding capacity is greater than 0.5mg per ml of c-myc agarose resin.</span></div><div class = "text-block"><span style = "font-weight:bold;">Reagent: </span><span>Anti-c-Myc Agarose Affinity Gel is supplied as a 50% suspension in phosphate-buffered saline, pH 7.2, containing 0.09% sodium azide.</span></div><div class = "text-block"><span style = "font-weight:bold;">Precautions and Disclaimer: </span><span>This product is for research use only. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.</span></div><div class = "text-block"><span style = "font-weight:bold;">Storage/Stability: </span><span>Anti-c-Myc Agarose Affinity Gel should be stored between 2°C and 8°C for maximum stability. The unopened product is stable for one year when stored as indicated. After use, the resin should be regenerated and stored in TBS or PBS buffer (pH 7.2) containing 0.09% sodium azide.</span></div></div>

[STEPS]
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from crude E. coli extracts]
Grow the bacteria cells under the condition that induce production of c-Myc fusion proteins.
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from crude E. coli extracts]
Harvest the cells by centrifugation at 10,000 rpm for 20 minutes at 4°C.
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from crude E. coli extracts]
Decant the medium and freeze the cell pellet at -20°C for enhanced cell lysis. At this step, cell pellet can be stored at - 80°C for up to 2 years.
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from crude E. coli extracts]
Thaw the cell pellet at 37°C. Mix pellet in appropriate amount of Breakage Buffer then proceed to lyse cells by sonication. Nuclease treatment is optional.
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from crude E. coli extracts]
Centrifuge at 10,000 rpm for 30 minutes.Note: If the fusion protein is soluble, discard pellet of cell debris. Transfer the supernatant into a clean container and filter with a 0.45 or 0.22µm filter to remove particulate matter. Supernatant is now ready for c-Myc column purification.If the fusion protein is in insoluble form, solublize the inclusion body after centrifugation by denaturant, refold the protein properly, change buffer into PBS (pH 7.2) then filter the refolded protein sample by a 0.45 or 0.22µm filter to be ready for purification.
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from mammalian cells]
Adherent cells: trypsinize cells. Suspension cells: skip this step.
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from mammalian cells]
Collect enough mammalian cells by centrifugation.
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from mammalian cells]
Properly lyse the cell pellet.
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from mammalian cells]
Centrifuge to remove the cell debris.
?. [Part I. Cell Lysate Preparation: Purifying c-Myc fusion proteins from mammalian cells]
Supernatant should be filtered by a 0.45 or 0.22µm filter to prepare for c-Myc column purification.
?. [Part II. Resin Preparation]
Thoroughly suspend the resin by gentle inversion until the Anti-c-Myc-Agarose Affinity Gel is a uniform suspension of gel beads. Transfer an appropriate aliquot of gel slurry into a clean chromatography column and allow the gel bed to drain by gravity. Do not let the gel bed run dry.
?. [Part II. Resin Preparation]
Equilibrate the column by two sequential column volumes of PBS containing 0.5M NaCl.
?. [Part III. c-Myc Fusion Protein Purification]
For smaller volume of cell lysate (100~200ml), pass the cell lysate through the affinity gel column once or multiple times to enhance the binding efficiency. For large volume of cell lysate, add the equilibrated c-Myc resin to the cell lysate and mix gently at room temperature or 4°C for 3 to 5 hours using an overhead mixing device or a stirrer. Then load sample mixture onto a clean chromatography column, allow the resin to settle and drain by gravity. Collect the flow through and test on a SDS-gel to confirm the complete binding of fusion protein to the c-Myc resin. (For very small amount of protein extract (1~2ml), add equilibrated c-Myc resin to the individual tubes with cell extract and gently rock at room temperature or 4°C for 3-5 hours, followed by centrifugation at 2,000 rpm for 2-3 minutes).
?. [Part III. c-Myc Fusion Protein Purification]
Wash the column by equilibration buffer. Monitor the flow through by Bradford assay. Wash appropriate amount of equilibration buffer until the Bradford assay indicates baseline is met.
?. [Part III. c-Myc Fusion Protein Purification]
Elute the protein by Acid Elution Buffer. Elute into fractions containing appropriate amount of 2M Tris pH 8.0 to immediately neutralize the elute (the exact amount of 2M Tris pH 8.0 should be determined before experiment by testing the pH value of various volume combination of acid elution buffer and 2M Tris pH 8.0). Monitor the eluted protein concentration in the sequential elute aliquots by Bradford Assay or optical absorbance at 280nm. Terminate elution step when baseline is met. Alternatively, the fusion protein can be eluted by adding c-Myc peptide to compete the binding to the affinity resin.
?. [Part III. c-Myc Fusion Protein Purification]
Run a SDS-gel for all the elute fractions and combine the ones with desired concentration and purity base on the gel result. Dialyze the protein product into appropriate buffer and stored properly.
?. [Part IV. c-Myc Affinity Gel Regeneration and Storage]
Wash the resin with at least five column volume of high-salt buffer to remove any residual protein retained on the resin after elution.
?. [Part IV. c-Myc Affinity Gel Regeneration and Storage]
Re-equilibrate the resin with at least five column volumes of PBS/0.5M NaCl for immediate usage. If not used immediately, wash the resin by two column volumes of PBS, then store in sufficient amount of PBS/0.09% azide at 4°C. The resin can be used for up to 10 times without any obvious loss of binding capacity.