Immunoprecipitation Kit -DYKDDDDK(Flag®)Tag Immunomagnetic Beads

Cat: TB101274

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Product Content
 
Contents TB101274-20 TB101274-100 Storage
DYKDDDDK Tag Immunomagnetic Beads1 1 mL 5 mL 2-8℃ for 12 months
NP40 Cell Lysis Buffer2 4 mL 22 mL -20℃ for 12 months
5×TBST(pH7.4) Required but not supplied  
1×TBST(pH7.4) Required but not supplied  
ddH2O Required but not supplied  
DYKDDDDK Tag Positive Cell Lysis 300 μg 300 μg -20℃ for 12 months
Alkaline Elution Buffer 3 mL 15 mL 2-8℃ for 12 months
Acidity Elution Buffer 3 mL 15 mL 2-8℃ for 12 months
Neutralization Buffer 2 mL 8 mL 2-8℃ for 12 months
DYKDDDDK Synthetic Peptide Not included (refer related product PP101274) Not included (refer related product PP101274) -20℃ for 12 months

[1] DYKDDDDK Tag Immunomagnetic Beads contain immunomagnetic beads (2 mg/mL) in phosphate buffered saline (PBS, pH 7.4) with sodium azide (0.1%).
[2] Using NP-40 cell lysate buffer in the kit is required,otherwise,the magnetic beads may be precipitated.

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Product Description
 

The DYKDDDDK Tag Immunomagnetic Beads, conjugated with DYKDDDDK Antibody (101274-MM13), are used for immuneprecipitation (IP) of DYKDDDDK-tagged proteins expressed in vitro expression systems. For IP, the Immunomagnetic Beads are added to a sample containing DYKDDDDK-tagged proteins to form an Immunomagnetic Beads-protein complex. The complex is removed from the solution manually against a Magnetic Separator. The bound DYKDDDDK-tagged proteins are dissociated from the Immunomagnetic Beads using an Elution Buffer.

Antibody Information
 

Antibody: DYKDDDDK (FLAG® epitope Tag) Antibody, Mouse Mab (101274-MM13)
Immunogen: A synthetic peptide corresponding to the DYKDDDDK-tag sequence (CDYKDDDDK).
Clone ID: MM13
Isotype: lgG1
Specificity: Recognize N-terminal and C-terminal Flag Tag in fusion proteins.
Preparation: This antibody was produced from a hybridoma resulting from the fusion of a mouse myeloma with B cells obtained from a mouse immunized with purified, the synthetic peptide corresponding to the DYKDDDDK-tag sequence (CDYKDDDDK). The IgG fraction of the cell culture supernatant was purified by Protein A affinity chromatography.

IP Experimental results
 
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Items Lane

Sample (30 μg)

(Whole cell lysate)

A B
pSTEP2 Transfected 293 Flag-mFABP4-GST
Beads SBI Anti-DYKDDDDK (FLAG® epitope Tag) Immunomagnetic Beads-30 μL
WB detection antibody Anti-DYKDDDDK (FLAG® epitope Tag) Antibody, Mouse MAb (101274-MM13) at 10 μg/mL
Gel 13% SDS-PAGE reducing gel
Secondary antibody Dylight 800-labeled antibody to mouse IgG (H+L), at 1:5000 dilution
Protocol
 
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Fig. 1 Immunoprecipitation (IP) Protocol

The protocol (Fig. 1) uses 50 μL flag Tag Immunomagnetic Beads, but this can be scaled up or down as required.
Cell Lysis
Cells may be lysed using any standard cell lysis protocol in accordance with your starting materials. We suggest using NP40 Cell Lysis Buffer (supplied with kit). Add prtease inhibitor (such as PMSF at 1mM) if needed.
Immunoprecipitate Target Antigen
1. Add 50 μL of Immunomagnetic Beads into a 1.5 mL microcentrifuge tube.
2. Add 150 μL of 1× TBST buffer to the Immunomagnetic Beads and gently vortex to mix.
3. Place the tube into a Magnetic Separator to collect the Immunomagnetic Beads against the side wall of the tube. Remove and discard the supernatant.
4. Add 1 mL of 1×TBST buffer to the tube. Invert the tube several times or gently vortex to mix for 1 min. Collect the Immunomagnetic Beads with a Magnetic Separator. Remove and discard the supernatant.
5. Add the sample containing target protein (~100 μg of protein in 100 μL) to the pre-washed Immunomagnetic Beads, add 400 μL of 1×TBST buffer and incubate at room temperature for 30 min with mixing.
6. Collect the Immunomagnetic Beads with a Magnetic Separator , remove the unbounded sample and save for analysis.
7. Add 300 μL of 5× TBST buffer to the tube and gently mix. Collect the Immunomagnetic Beads and discard the supernatant. Repeat this wash twice.
8. Add 300 μL of ddH2O to the tube and gently mix. Collect the Beads on a Magnetic Separator and discard the supernatant.
Elute Target Antigen
A. Neutral Elution Protocol
1. Prepare DYKDDDDK peptide (PP101274) at 1mg/mL in PBS.
2. Add 50 μL 1 mg/mL DYKDDDDK peptide to the Immunomagnetic Beads, gently vortex to mix and incubate the sample at 37 ℃ on a rotator for 5-10 min. Elution may be performed at reduced temperatures, but lower yields may result.
3. Separate the Immunomagnetic beads on a Magnetic Separator and save the supernatant containing the target antigen.
4. Repeat Elution step once for higher recovery.
B. Alkaline Elution Protocol
1. Add 100 μL of Alkaline Elution Buffer to the tube.
2. Gently vortex to mix and incubate the sample at room temperature on a rotator for 5 min.
3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the target antigen.
4. To neutralize the sample, add 50 μL of Neutralization Buffer for each 100 μL of eluate.
C. Acidity Elution Protocol
1. Add 100 μL Acidity Elution Buffer.
2. Gently vortex to mix and incubate the sample at room temperature on a rotator for 5-10 min.
3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the target antigen.
4. To neutralize the low pH, add 50 μL of Neutralization Buffer for each 100 μL of eluate.
D. Elution Using Sample Buffer
1. Add 100 μL of SDS-PAGE Sample Buffer to the tube.
2. Gently vortex to mix and incubate the sample at 95-100℃ for 5-10 min.
3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the antigen.
Usage of positive cell lysate
When detecting the antigen with western blotting, the positive cell lysates (supplied in the KIT) can be used as a positive western blotting cell lysates sample.
Note: For tag magnetic beads the positive cell lysates also can be used as a IP cell lysates sample.

Reference Information
 

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Trouble Shooting

Problem Possible Cause Solution
Little or no DYKDDDDK-tagged protein is detected Tagged protein degraded Include protease inhibitors (PMSF) in the lysis buffer
Use new lysate or lysate stored at -80°C
No or minimal tagged protein was expressed Verify protein expression by SDS-PAGE or Western blot
analysis of the lysate using an DYKDDDDK-tagged positive control as a reference
Increase the amount of lysate used for IP/Co-IP
Use a more sensitive detection system
Magnetic Beads aggregated Magnetic Beads were frozen or centrifuged Handle the Beads as directed in the instructions
Buffer was incompatible with magnetic beads
Detergent was not added to the wash and bind solutions
Failure to co-IP interacting protein Wash conditions were too stringent for the weak or transient interaction Reduce the number of washes
Lower the ionic strength of the wash buffer
Interacting protein was expressed at a low level Apply additional protein sample
Use a more sensitive detection systemds
Buffer system was not optimal for the protein: protein interaction Optimize the co-IP buffer
Insufficient sample was loaded on the gel for Western blot detection Elute sample in 30% acetonitrile 0.5% formic acid, then
Bring the sample back up in SDS- PAGE Sample Buffer and load entire elution fraction on

Other Proteins

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