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Anti-DYKDDDDK tag (L5) Affinity Gel

互联网2013-11-13

840

实验试剂

Column equilibration buffer:

1xPBS/0.5M NaCl

High salt wash buffer:

50mM Tris 8.0/2M NaCl

Elution buffer:

200mM Glycine/150mM NaCl, PH2.2

Neutralization buffer:

Bacteria breakage buffer (depend on the laboratory preference):

50 mM Tris PH 8.0/150mM NaCl, 1mM EDTA, 1mM DTT, 1mM PMSF and 1mg/ml lysozyme.

Mammalian breakage buffer (depend on the laboratory preference):

1xPBS or 50mM Tris 7.4/ 150mM NaCl supplemented with 1% NP40.

0.25% Na-deoxycholate.

1mM EDTA with 1mM PMSF and protease inhibitor cocktail.

实验步骤

Part I. Cell Lysate Preparation

1. Purifying DYKDDDDK-tag fusion proteins from crude E.coli extracts.

1) Grow the bacteria cells under the condition that induce production of DYKDDDDK-tag fusion proteins.

2) Harvest the cells by centrifugation at 10,000 rpm for 20 minutes at 4°C.

3) 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.

4) Thaw the cell pellet at 37°C. Mix in appropriate amount of Breakage Buffer.

5) Sonicate mixture to completely lyse cells.

6) Centrifuge at 10,000 rpm for 30 minutes. Transfer supernatant into a clean container and filtered with a 0.45 or 0.22µm filter.

7) If the DYKDDDDK-tag fusion protein is in soluble form, this supernatant is ready for L5 column purification. If the DYKDDDDK-tag fusion protein is in an insoluble form, solubilize the inclusion body after centrifugation step (Step 6), refold the protein properly, change buffer into PBS (PH7.2) and filter the protein sample by a 0.45 or 0.22 µm filter to be ready for L5 purification.

2. Purifying DYKDDDDK-tag fusion proteins from mammalian cells

1) Adherent cells: trypsinize cells.Suspension cells: skip this step.

2) Collect enough mammalian cells by centrifugation.

3) Properly lyse the cell pellet.

4) Centrifuge to remove the cell debris.

5) Supernatant should be filtered by a 0.45 or 0.22µm filter to prepare for L5 purification.

Part II. Resin Preparation

1. Thoroughly suspend the resin by gentle inversion until the bottle of Anti- DYKDDDDK-tag (L5) affinity gel is a uniform suspension of gel beads. Transfer an appropriate aliquot into a clean chromatography column and allow the gel bed to drain naturally. Do not let the gel bed run dry.

2. Equilibrate the column by two sequential column volumes of PBS containing 0.5M NaCl.

Part III. L5 column purification

1. For smaller volume of cell lysate (100~200 ml), pass the cell lysate through the DYKDDDDK-tag (L5) affinity gel column once or multiple times to enhance the binding efficiency. For large volume of cell lysate, add the equilibrated L5 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 platform shaker. Then load sample mixture onto a clean chromatography column, allow the resin to settle and drain naturally. Collect the flow through and test on a SDS-gel to confirm the complete binding of DYKDDDDK-tag fusion protein to the L5 resin. For very small amount of protein extract (1~2 ml), add equilibrated L5 resin (binding capacity is >0.8 mg/ml) to the individual tubes and gently rock at room temperature or 4°C for 3-5 hours, then proceed with the following steps in the tube be table-top centrifugation at 2,000 rpm for 2-3 minutes at each step to re-settle the resin and re-collect the supernatant. (ie. Immunoprecipitation of the DYKDDDDK-tag fusion protein).

2. Wash the column by equilibration buffer (PBS/0.5M NaCl). Monitor the flow through by BFA reaction. Wash appropriate amount until the BFA test becomes baseline.

3. Elute the protein by Acid Elution Buffer (200mM Glycine/150mM NaCl, PH2.2). Elute into aliquots containing appropriate amount of 2M Tris PH8.0 to immediately neutralize the elute (the exact amount of 2M Tris PH8.0 should be determined before experiment by testing the PH value of various combination of acid elution buffer and2M Tris PH8.0 ). Monitor the protein concentration in the sequential elute aliquots by BFA reaction or A280. Terminate elution step when protein is completely eluted. Alternatively, the fusion protein can be eluted by adding DYKDDDDK peptide to compete the binding to the L5 resin.

4. Run a SDS-gel for all the elute aliquots 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. L5 Resin Regeneration and Storage

1. Wash the resin with at least five column volume of high-salt buffer (50mM Tris 8.0/2M NaCl) to remove any residual protein retained on the resin.

2. 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 stored 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.

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