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抗人颗粒酶B/Granzyme| 货号： | TB100028-1 |
| 规格： | 1mL |
| Contents | TB100028-1 | TB100028-5 | Storage |
| HA Tag Immunomagnetic Beads1 | 1 mL | 5 mL | 2-8℃ for 12 months |
| NP40 Cell Lysis Buffer | 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 | ||
| HA 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 |
| Magnetic Separator | Not included (refer related product MAGS001) | One MAGS001 included in China2 | |
| HA Synthetic Peptide | Not included (refer related product PP100028) | Not included (refer related product PP100028) | -20℃ for 12 months |
[1] HA Tag Immunomagnetic Beads contain immunomagnetic beads (2 mg/mL) in phosphate buffered saline (PBS, pH 7.4) with sodium azide (0.1%).
[2] The Magnetic Separator cannot be included for oversea customers due to shipment prohibition.
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The HA Tag Immunomagnetic Beads, conjugated with HA tag Antibody (100028-MM15), are used for Immuneprecipitation / IP of HA-tagged proteins expressed in vitro expression systems. For IP, the Immunomagnetic Beads are added to a sample containing HA-tagged proteins to form a bead-protein complex. The complex is removed from the solution manually against a Magnetic Separator. The bound HA-tagged proteins are dissociated from the Immunomagnetic Beads using an Elution Buffer.
Antibody: HA Tag Antibody, Mouse MAb (100028-MM15)
Immunogen: A synthetic peptide corresponding to the HA-tag sequence.
Clone ID: MM15
Isotype: IgG
Specificity: Recognize N-terminal and C-terminal HA 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, a synthetic peptide corresponding to the HA-tag sequence. The IgG fraction of the cell culture supernatant was purified by Protein A affinity chromatography.
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| Items | Lane | ||
| Sample (30 μg) (Whole cell lysate) | A | B | C |
| HA-ARG1-myc Transfected 293 | myc-ARG1-HA Transfected 293 | pSTEP2 Transfected 293 | |
| Beads | SBI Anti-HA Tag Immunomagnetic Beads-30 μL | ||
| WB detection antibody | Anti-HA Tag Antibody, Mouse Mab (100028-MM10) at 1 μg/mL | ||
| Gel | 13% SDS-PAGE reducing gel | ||
| Secondary antibody | Dylight 800-labeled antibody to mouse IgG (H+L), at 1:5000 dilution | ||
|
| Fig. 1 Immunoprecipitation (IP) Protocol |
The protocol (Fig. 1) uses 50 μL HA 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).
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 HA peptide (PP100028) at 1mg/mL in PBS.
2. Add 50 μL 1 mg/mL HA 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 15 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.
Related Products
| Products | Cat No. |
| Magnetic Separator-1.5 (2 tubes) for IP | MAGS001 |
| HA Synthetic Peptide | PP100028 |
| Immunoprecipitation Kit -Immunomagnetic Beads Protein A | BA10600 |
| Immunoprecipitation Kit -Immunomagnetic Beads Protein G | BG13103 |
| Immunoprecipitation Kit -Immunomagnetic Beads Protein L | BL11044 |
| Immunoprecipitation Kit -MYC Tag Immunomagnetic Beads | TB100029 |
| Immunoprecipitation Kit -GST Tag Immunomagnetic Beads | TB11213 |
| Immunoprecipitation Kit -GFP Tag Immunomagnetic Beads | TB13105 |
| Immunoprecipitation Kit -V5 Tag Immunomagnetic Beads | TB100378 |
| Immunoprecipitation Kit -DYKDDDDK（Flag®）Tag Immunomagnetic Beads | TB101274 |
Trouble Shooting
| Problem | Possible Cause | Solution |
| Little or no HA-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 HA-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 |
