ESO wikiadmin at 14:48, 30 May 2025

2025-05-30T14:48:17Z

← Older revision		Revision as of 16:48, 30 May 2025
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	=== Procedure ===		== Procedure ==
	~~----~~

	==== Equilibration of beads ====		==== Equilibration of beads ====

ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials ! |- |Recombinant protein |Bait protein TwinStrepTagII tagged protein, target proteins (HisSUMO or GST tagged protein of interest) |- |Pull down buffer |50 mM Tris/Cl pH 8, 150 mM NaCl, 10% Glycerol, 0.05% Tween. Optional: 10 µM ZnCl₂ in case of Zinc- finger proteins or 2 mM MgCl₂, CaCl₂ or MnCl₂ in case of metalloproteins) |- |Elution buffer EXT |50 mM Tris/Cl pH 8, 150 mM NaCl, 50 mM Biotin..."

2025-02-27T12:31:28Z

Created page with "{| class="wikitable" |+ !Materials ! |- |Recombinant protein |Bait protein TwinStrepTagII tagged protein, target proteins (HisSUMO or GST tagged protein of interest) |- |Pull down buffer |50 mM Tris/Cl pH 8, 150 mM NaCl, 10% Glycerol, 0.05% Tween. Optional: 10 µM ZnCl2 in case of Zinc- finger proteins or 2 mM MgCl2, CaCl2 or MnCl2 in case of metalloproteins) |- |Elution buffer EXT |50 mM Tris/Cl pH 8, 150 mM NaCl, 50 mM Biotin..."

New page

{| class="wikitable"
|+
!Materials
!
|-
|Recombinant protein
|Bait protein TwinStrepTagII tagged protein, target proteins (HisSUMO or GST tagged protein of interest)
|-
|Pull down buffer
|50 mM Tris/Cl pH 8, 150 mM NaCl, 10% Glycerol, 0.05% Tween. Optional: 10 µM ZnCl2 in case of Zinc- finger proteins or 2 mM MgCl2, CaCl2 or MnCl2 in case of metalloproteins)
|-
|Elution buffer EXT
|50 mM Tris/Cl pH 8, 150 mM NaCl, 50 mM Biotin) '''store dark and cold'''
|-
|
|StrepTactin Sepharose Beads
|}

=== Procedure ===
----

==== Equilibration of beads ====

* Use 10 µl of StrepTactin-Sepharose beads per sample and transfer into fresh 1.5 ml Eppendorf tube. Add 200 µl pulldown buffer
* Centrifuge at 2700 rcf for 2 min. Remove supernatant
* Equilibrate beads by washing them twice in 200 µl pulldown buffer. Remove supernatant after last washing step

==== Preparation of bait and target protein ====

* Centrifuge protein extract prior to pulldown 20 min at 10.000 g to remove any debris or aggregated protein.
* Prepare 130 µl of bait protein (TwinStrepTag-proteinX) solution by taking 2.5 µg of bait protein and adjust with pulldown buffer to a final volume of 130 µl. Take 30 µl of solution, mix with 10 µl 4xSDS and boil for 5 min at 95°C. Refer to as “Input”
* Prepare 130 µl of target protein (eg. HisSUMO-proteinY) solution by taking 5-10 µg of target protein and adjust with pulldown buffer to final volume of 130 µl. Take 30 µl of solution, mix with 10 µl 4xSDS and boil for 5 min at 95°C. Refer to as “Input”

==== Loading of beads ====

* Resuspend equilibrated sepharose beads in 100 µl bait protein solution
* Incubate 30 min at room temperature on head to head rotor
* Centrifuge at 2700 rcf for 2 min and remove supernatant (take 30 µl of supernatant and mix with 4xSDS. Boil 5 min at 95°C. Refer to as “unbound” fraction)
* Wash 3x with 100 µl pulldown buffer, mix by harsh shaking and centrifuge (2700 rcf, 2 min)

==== Pulldown ====

* Resuspend loaded beads in 100 µl target protein solution
* Incubate 2 h at 4° on head to head rotor
* Centrifuge (2700 rcf, 2min) and remove supernatant (take 30 µl of supernatant and mix with 4xSDS. Boil 5 min at 95°C. Refer to as “unbound” fraction)
* Wash 3x with 100 µl pulldown buffer, mix by harsh shaking and centrifuge (2700 rcf, 2 min)
* Elute under '''NATIVE''' conditions by adding 30 µl elution buffer BXT
* Incubate 10 min at room temperature on head to head rotor
* You may repeat this step to increase the yield (but not the concentration)
* Mix eluate with appropriate amount of 4xSDS and boil 5 min at 95°C

StrepTactin-Pulldown - Revision history

ESO wikiadmin at 14:48, 30 May 2025