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Immunoprecipitation/ IP Kit - Anti-Ephrin-B1 / EFNB1 Immunomagnetic Beads

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Immunoprecipitation/ IP Kit- Anti-Ephrin-B1 / EFNB1 Immunomagnetic Beads
Product Contents
ContentsPackage 1Package 2Storage
Anti-Ephrin-B1/EFNB1 Immunomagnetic Beads1 31 mL5 mL2-8℃ for 12 months
NP40 Cell Lysis Buffer4 mL22 mL-20℃ for 12 months
5×TBST(pH7.4)Required but not supplied
1×TBST(pH7.4)Required but not supplied
ddH2ORequired but not supplied
Ephrin-B1/EFNB1 Positive Cell Lysis300 μg300 μg-20℃ for 12 months
Alkaline Elution Buffer3 mL15 mL2-8℃ for 12 months
Acidity Elution Buffer3 mL15 mL2-8℃ for 12 months
Neutralization Buffer2 mL8 mL2-8℃ for 12 months
Magnetic Separator Not included (refer related product MAGS001)One MAGS001 included in China2

[1] The IP KIT contains anti-Ephrin-B1/EFNB1 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.

[3] Shipping: Immunomagnetic Beads kits are shipped at ambient temperature in which immunomagnetic beads are provided in liquid buffer.

Product Description

The Anti-Ephrin-B1/EFNB1 Immunomagnetic Beads, conjugated with Anti-Ephrin-B1/EFNB1 antibody, are used for immuneprecipitation (IP) of Ephrin-B1/EFNB1 proteins which expressed in vitro expression systems and bacterial and mammalian cell lysates. For IP, the beads are added to a sample containing Ephrin-B1/EFNB1 proteins to form a bead-protein complex. The complex is removed from the solution manually using a Magnetic Separator. The bound Ephrin-B1/EFNB1 proteins are dissociated from the Immunomagnetic Beads using an elution buffer.

Antibody Information
Antibody:80106-T52
Immunogen:Recombinant Rat Ephrin-B1 / EFNB1 protein (Catalog#80106-R08H)
Isotype:Rabbit IgG
Clone ID:
Molecule:EFNB1
抗體製備方法:Produced in rabbits immunized with purified, recombinant Rat Ephrin-B1 / EFNB1 (rh Ephrin-B1 / EFNB1; Catalog#80106-R08H; P52796; Met1-Thr229). Ephrin-B1 / EFNB1 specific IgG was purified by Rat Ephrin-B1 / EFNB1 affinity chromatography.
特異性:Rat Ephrin-B1 / EFNB1
Guaranteed Applications:IP,Minimum Protein Purification
IP Protocol

Fig. 1 Immunoprecipitation (IP) Protocol

The protocol (Fig. 1) uses 50 μL Anti-Ephrin-B1/EFNB1 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 beads against the wall side 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 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 Immunomagnetic Beads on a Magnetic Separator and discard the supernatant.
Elute Target Antigen.
A. Alkaline Elution Protocols
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.
B. Acidity Elution
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.
C. 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 95-100⁰C for 5-10 min.
3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the antigen.

Reference Information

Related product

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Immunoprecipitation Kit -Immunomagnetic Beads Protein L KitBL11044
Immunoprecipitation Kit -Anti-DYKDDDDK(Flag®) Tag Immunomagnetic Beads Kit TB101274
Immunoprecipitation Kit -Anti-GFP Tag Immunomagnetic Beads Kit TB13105
Immunoprecipitation Kit -Anti-Myc Tag Immunomagnetic Beads Kit TB100029
Immunoprecipitation Kit -Anti-HA Tag Immunomagnetic Beads Kit TB100028
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Immunoprecipitation Kit -Anti-GST Tag Immunomagnetic Beads Kit TB11213

Trouble Shooting

ProblemPossible CauseSolution
Little or no protein is detectedProtein degradedInclude protease inhibitors (PMSF) in the lysis buffer
Use new lysate or lysate stored at -80℃
No or minimal protein was expressedVerify protein expression by SDS-PAGE or Western blot
analysis of the lysate using an positive control as a reference
Increase the amount of lysate used for IP/Co-IP
Use a more sensitive detection system
Immunomagnetic Beads aggregatedImmunomagnetic beads were frozen or centrifugedHandle the Immunomagnetic Beads as directed in the instructions
Buffer was incompatible with Immunomagnetic Beads
Detergent was not added to the wash and bind solutions
Failure to co-IP interacting proteinWash conditions were too stringent for the weak or transient interactionReduce the number of washes
Lower the ionic strength of the wash buffer
Interacting protein was expressed at a low levelApply additional protein sample
Use a more sensitive detection system
Buffer system was not optimal for the protein: protein interactionOptimize the co-IP buffer
Insufficient sample was loaded on the gel for Western blot detectionElute 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
Ephrin-B1/EFNB1 研究背景

Ephrin-B1 also known as EFNB1, is a member of the ephrin family. The transmembrane- associated ephrin ligands and their Eph family of receptor tyrosine kinases are expressed by cells of the SVZ. Eph/ephrin interactions are implicated in axon guidance, neural crest cell migration, establishment of segmental boundaries, and formation of angiogenic capillary plexi. Eph receptors and ephrins are divided into two subclasses, A and B, based on binding specificities. Ephrin subclasses are further distinguished by their mode of attachment to the plasma membrane: ephrin-A ligands bind EphA receptors and are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) linkage, whereas ephrin-B ligands bind EphB receptors and are anchored via a transmembrane domain. An exception is the EphA4 receptor, which binds both subclasses of ephrins. EphrinB1 and B class Eph receptors provide positional cues required for the normal morphogenesis of skeletal elements. Another malformation, preaxial polydactyly, was exclusively seen in heterozygous females in which expression of the X-linked ephrinB1 gene was mosaic, so that ectopic EphB-ephrinB1 interactions led to restricted cell movements and the bifurcation of digital rays.

Rat Ephrin-B1/EFNB1 參考資料
  • Davy A, et al. (2004) Ephrin-B1 forward and reverse signaling are required during mouse development. Genes Dev. 18(5): 572-83.
  • Compagni A, et al. (2003) Control of skeletal patterning by ephrinB1-EphB interactions. Dev Cell. 5(2): 217-30.
  • Wieland I, et al. (2004) Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome. Am J Hum Genet. 74(6): 1209-15.
  • Size / Price
    貨號: MB80106-T52-1
    目錄價: 
    單價:      (You Save: )
    規格:
    1 mL
    5 mL
    數量:+-
    庫存In Stock
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