MagpoinsTM His-Tag Immunoprecipitation KIT

Temporarily not available outside of China.
Product Content
 
Contents TBN001-20 TBN001-100 Storage
MagpoinsTM His-Tag 1 mL 5 mL 4 ℃, 12 month
NP40 cell lysate buffer 4 mL 20 mL 4 ℃, 12 month
2×Binding/Wash Buffer 30 mL 160 mL 4 ℃, 12 month
His Elution Buffer 3 mL 15 mL 4 ℃, 12 month
2×Pull-down Buffer 30 mL 160 mL 4 ℃, 12 month
Magnetic Separator 1 each 1 each RT

MagpoinsTM His-Tag Immunoprecipitation Kit contains 40 mg beads/mL in 20% ethanol and has a capacity of 2mg/mL his-tag protein (28kDa).

Product Description
 

MagpoinsTM His-tag IP KIT was developed for the isolation of his-tag proteins. These MagpoinsTM are coated in a nickel-based Immobilized Metal beads. The priority of beads bind his tag proteins is higher selectivity compared to Agarose and Sepharose based bead systems.

MagpoinsTM makes the purification of protein more quickly and easily:

Add the beads to a sample containing his-tag proteins and allow the proteins to bind to the MagpoinsTM. Isolated proteins can be left on the beads and used directly in downstream applications. Alternatively, the isolated his-tag protein can be eluted from the beads. The elution conditions are less stringent than other technologies thus yielding more functional isolated proteins. These characteristics make MagpoinsTM His-Tag Immunoprecipitation Kit the ideal product for purifying his-tag proteins expressed in E. coli, mammalian cell, yeast and so on.

Preparation of cell lysates
 

There are many different ways for preparing a cell lysate containing expressed his-tag proteins. It is important that the lysate does not contain EDTA (or other chelators), ionic detergents, DTT or DTE. A pH between 7 and 8 should be used.

a. The NP40 cell lysis buffer for mammalian and insect cells (supplied with this KIT). See the cell lysis protocol on page 3.
b. French press
c. Sonication

Efficiency of lysis can be increased by the addition of lysozyme (To avoid a sticky pellet, add SuperNuclease (Cat#: SSNP01) In order to avoid protein degradation add Power Protease Inhibitor.

We recommend using the standard protocol for different lysate methods.

Immunoprecipitation Protocol
 

Prepare your sample containing his-tag protein. Preferentially, you may prepare your sample in a total volume of 700 μL 1×Binding/Wash Buffer.

1. Thoroughly resuspend the MagpoinsTM in a tube (vortex >30 sec or tilt and rotate 5 min).
2. Transfer 50 μL (2 mg) MagpoinsTM to a micro-centrifuge tube. Place the tube on a magnet separator (Cat#: MAGS001) for 1 min. Aspirate and discard the supernatant. Add your sample (prepared in Binding/Wash Buffer) to beads, Mix well.
3. Incubate on a roller for 5 min at room temperature (or colder if the protein is unstable at room temperature). The incubation time may be increased with the incubate temperature lowing.
4. Place the tube on the magnet separator for 1 min, then discard the supernatant.
5. Wash the beads 4 times with 300 μL Binding/Wash Buffer by placing the tube on a magnet separator for 1 min and discard the supernatant. Resuspend the MagpoinsTM thoroughly between each washing step.
6. If the protein is to be eluted, proceed to step 7. If you wish to continue with a Pull-down, then continue to step 1 in "Protein Pull-down".
7. Add 100 μL His-Elution Buffer. Incubate the suspension on a roller for 5 min at room temperature (or colder if the protein is unstable at room temperature).
8. Put on the magnet for 1 min and transfer the supernatant containing the eluted histidine-tagged protein to a clean tube.

Protein Pull-down
 

1. Prepare your sample in Pull-down buffer (350 μL) in a total volume of up to 700 μL.
2. Add your sample (prepared in Pull-down Buffer) to the bead/protein complex from step 5 in "Immunoprecipitation Protocol".
3. Incubate on a roller for 10 min at room temperature (or cold if the protein is unstable at room temperature). The incubation time may be increased with the temperature lowing.
4. Place the tube on a magnet separator for 1 min, then discard the supernatant.
5. Wash the beads 4 times with 300 μL Binding/Wash Buffer by placing the tube on a magnet separator for 1 min and discard the supernatant. Resuspend the MagpoinsTM thoroughly between each washing step.
6. Add 100 μL His-Elution Buffer. Incubate the suspension on a roller for 5 min at room temperature (or cold if protein is unstable at room temperature).
7. Collect the MagpoinsTM at the tube wall using a magnet separator and transfer the supernatant containing the eluted his-tag protein and its interacting protein to a clean tube. The elution volume may be decreased to 50 μL.

Cell Lysis Protocol
 

A:Lysing Cells Grown as Monolayer Cultures

1. Discard the culture medium. Wash cells twice with ice-cold PBS. Place the culture dishes on ice.
2. Add 1.0 mL of the NP40 buffer of choice (pre-cold to 4°C) per 100 mm dish.
For culture dishes of other sizes, adjust the volume of lysis buffer accordingly.
3. Incubate the cells for 10-30 min (depending on the cell line being studied) on ice. Rock the dishes occasionally. 4. Collecting cells in a tube on the bed of ice, allowing the NP40 buffer to drain to one side. Remove the lysate with a pipette. Transfer to a micro-centrifuge tube (or other suitable centrifuge tube). Repeat with each of the remaining dishes.
5. Centrifuge the lysate at 12,000 g for 10 min at 4 °C.
6. Carefully remove the supernatant to a fresh tube, making sure not to disturb the pellet. Store the lysate at -20℃ until it is needed.

B: Lysing Cells Grown in Suspension

1. Harvest the cells by centrifugation at 480 g for 10 min. Decant and discard the supernatant.
2. Carefully, wash the cell pellet twice with ice-cold PBS. Place the washed cell pellet on ice.
3. Resuspend the pellet in 1.0 mL of the NP40 buffer of choice (pre-cold to 4 °C) per 1 × 107 to 5 × 107 cells. 4. Incubate the cells for 15 min on ice, vortex the tube occasionally.
5. Centrifuge the lysate at 12,000 g for 10 min at 4°C.
6. Carefully, remove the supernatant to a fresh tube, making sure not to disturb the pellet. Store the lysate at -20°C until it is needed.

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