ApaNovis PhageSelect 20 Kit

Transcription

1 ApaNovis PhageSelect 20 Kit M13 Bacteriophage Purification for Phage Display Cat. No AO For research use only. Store at RT. 1

2 ApaNovis PhageSelect 20 Kit 1. Features Efficient purification of M13 bacteriophage in phage display Fast procedure - less than 1 hour High yields - up to particles from 20 ml culture Standardization for phage display 2. Description Phage display is employed in the design of specific antibodies for therapeutic, diagnostic and molecular biology applications. The isolation of high-affinity phage encoding the displayed proteins/ peptides specific for a particular target usually involves 3-5 biopanning rounds in which target-specific phages are enriched by affinity selection. Each selection round comprises binding, elution and tedious purification of amplified phage. The conventional phage purification takes one day. However, employing the ApaNovis PhageSelect 20 Kit, the time required for purification is reduced to less than one hour. As a result both phage purification and a new biopanning round can be performed on the same day. The obtained phages are PEG-free, infectious and can be readily adjusted to the buffer suitable for biopanning or storage. Bacterial Culture Centrifugation Phage Suspension Conventional Method ApaNovis PhageSelect 20 > 5h 2x PEG Precipitation Centrifugation Affinity Chromatography Phage Concentrate < 1h Phage Concentrate Biopanning same day Biopanning next day 2

3 3. Components 5 PhageSelect 20 Membrane Adsorber Spin Columns 125 ml PhageSelect 20 Binding Buffer 250 ml PhageSelect 20 Washing Buffer 20 ml PhageSelect 20 Elution Buffer 5 PhageSelect kda Spin Columns 5 50 ml empty tubes for storage/sample dilution Storage Conditions Room temperature. Shipping Conditions Room temperature. Shelf Life 24 months from the date of purchase. 4. Additional Equipment Required Any centrifuge with a swing-out rotor that will accommodate 50 ml centrifuge tubes with conical bottom and can spin samples for up to 5,000 x g. 3

4 5. Purification Protocol 5.1 Growth Conditions Propagation of M13 bacteriophages is accomplished according to standard protocols in E. coli strain TG1 (Sambrook, J. et al.). Briefly, 200 µl of a fresh TG1 culture are diluted into 20 ml LB-medium and incubated at 37 C at 150 rpm until an OD550 of 0.5 is reached. Then M13 bacteriophages are added at a multiplicity of infection of approximately 10 and incubated at 37 C at 50 rpm for 30 min. After infection, bacteria are diluted 2:100 in 2xTY medium containing kanamycin at 50 µg/ml and the culture is incubated at 30 C at 150 rpm overnight (typically 12-14h). 5.2 Sample Preparation and Purification Spin 20 ml E. coli overnight cultures at 4,500 x g for 15 min to pellet bacteria. Prior to loading, equilibrate the PhageSelect 20 Membrane Adsorber Spin Column with 5 ml Binding Buffer. Spin for 5 min at 1,000 x g and discard the flow-through. 5.3 Sample Loading Transfer ml supernatant (containing M13 bacteriophages) to the PhageSelect 20 Membrane Adsorber Spin Column. Spin for 5 min at 1,000 x g. Wash the membrane with 18 ml Washing Buffer. Spin for 5 min at 1,000 x g and discard the flow-through. Repeat this step. 5.4 Elution of M13 Bacteriophages Add 3 ml of Elution Buffer to the PhageSelect 20 Membrane Adsorber Spin Column to elute the phages. Spin for 5 min at 500 x g. The flow-through contains the phages, which may already be used for the next panning round, if diluted into PBS or low-ionic strength buffers at desired ph. For standardization of your selection buffer, however, we recommend to perform steps Concentration and Buffer Exchange Transfer the eluate (3 ml) to the PhageSelect kda Spin Column. Add Binding Buffer up to 20 ml. Spin 45 min at 3,000 x g. Resuspend the concentrated phages by gently adding Binding Buffer or PBS. Determine phage titer (and go to the next panning round). Note: To avoid aggregation and loss of infectivity, do not reduce the volume to less than 0.5 ml; check the volume of the phage concentrate (in the upper chamber) at 30 min and centrifuge again, if necessary. 4

5 5.6 (Optional) Additional Washing Step In order to achieve higher sample purity of the phage for the next selection round, repeated washing of the phage can be conveniently performed on the PhageSelect kda Spin Column using Washing Buffer or PBS. 5.7 (Optional) Buffer Exchange after Elution In case you need a specific buffer for your phage in your biopanning application, buffer exchange can be conveniently performed using the PhageSelect kda Spin Column. To this end, replace the Binding Buffer (step 5.5) by your buffer of choice. Note: If you want to store M13 bacteriophages after concentration using the PhageSelect kda Spin Column in storage buffer containing glycerine, it is recommended to add glycerine directly to the phage concentrate rather than performing a buffer exchange with generic storage buffers for long time storage; glycerine-containing buffers take considerably longer to pass the ultracentrifugation device. 5

6 6. Literature Sambrook, J., Fritsch, E. F. & Maniatis, T. (1990) Molecular Cloning - A Laboratory Manual. Cold Spring Harbor, New York 7. Related Products Description Cat# Size / Phage Display optimized ER2738 Electrocompetent LU 12 transformations phage_display Cells (DUOS) > 2 x cfu/ug LU (6 x 50 ul) 24 transformations (12 x 50 ul) Phage Display optimized SS320 (MC1061 F ) Electrocompetent Cells (DUOS) > 4 x cfu/ug Phage Display optimized TG1 Electrocompetent Cells (DUOS) > 2 x cfu/ug Human Domain Antibody Phagemid Library LU 12 transformations phage_display (6 x 50 ul) LU 24 transformations (12 x 50 ul) LU 12 transformations phage_display (6 x 50 ul) LU 24 transformations (12 x 50 ul) DAB1000-GSL 1 Kit dab 6

7 8. Notes 7

8 BioCat products are intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes nor are they intended for use in humans. Products may not be resold, modified for resale, or used to manufacture commercial products without written approval of BioCat GmbH, Heidelberg. BioCat GmbH Im Neuenheimer Feld 584 D Heidelberg Tel.: Fax: