AM26519AF-N CD268 / BAFFR antibody

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0.1 mg / €350.00
Please visit the country specific website of Acris Antibodies or contact your local Distributor to buy this product.

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Mouse anti Human CD268 / BAFFR 8A7

AM26519AF-N

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  • AM26519AF-N

Product Description for CD268 / BAFFR

Mouse anti Human CD268 / BAFFR 8A7.
Presentation: Azide Free
Product is tested for Flow Cytometry, Paraffin Sections, Western blot / Immunoblot.

Properties for CD268 / BAFFR

Product Category Primary Antibodies
Target Category
Quantity 0.1 mg
Synonyms B cell-activating factor receptor, BAFF receptor, BAFF-R, BLyS receptor 3, BR3, TNFRSF13C, Tumor necrosis factor receptor superfamily member 13C
Presentation Azide Free
Reactivity Hu
Applications F, P, WB
Clonality Monoclonal
Clone 8A7
Host Mouse
Isotype IgG2a
Shipping to Worldwide
PDF datasheet View Datasheet
Manufacturer Acris Antibodies GmbH
Material safety datasheet MSDS for Monoclonal Antibodies (de)

Datasheet Extract

Immunogen
Swiss Prot Num:
Q96RJ3
Immunogen:
Human BAFF-R/BR3 transfectant
GeneID:
115650
Add. information

This product was originally produced by MBL International.

Application Western blot: 1 μg/ml.
Immunohistochemistry on paraffin sections: 5 μg/ml; Heat treatment is necessary; Microwave oven; 2 times for 10 minutes each in citrate buffer (pH 6.5).
Flow cytometry: 5 μg/mL (final concentration).
For details see protocols below.
Background

BAFF (B cell-activating factor belonging to the TNF family) is a membrane protein expressed by dendritic cells, monocytes, macrophages, follicular dendritic cells, activated T cells, activated neutrophils, and malignant B cells. BAFF, also known as BLyS (B lymphocyte stimulator), is a potent B cell growth factor. Proteolytic cleavage can result in the release of a soluble trimeric BAFF which binds to the BAFF-R/BR3, BCMA and TACI. BAFF-R/BR3 is the principal receptor for B cell survival and responses induced by BAFF.

Concentration 1.0 mg/ml
Protocols

SDS-PAGE & Western Blotting
1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) containing appropriate protease inhibitors. Incubate it at 4 oC with rotating for 30 minutes, then sonicate briefly (up to 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 oC and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the Lysis buffer to make an 8 mg/mL solution.
3) Mix the sample with an equal volume of Laemmli’s sample buffer.
4) Boil the samples for 2 minutes and centrifuge. Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis.
5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for specific transfer procedure.
6) To reduce nonspecific binding, soak the membrane in 10% Skimmed Milk in PBS for 1 hour at room temperature, or overnight at 4 oC.
7) Incubate the membrane with the anti-human BAFF-R/BR3 monoclonal antibody (8A7) diluted with PBS, pH7.2 containing 1% Skimmed Milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The optimal antibody concentration will depend on the experimental conditions.)
8) Wash the membrane with PBS (5 minutes x 6 times).
9) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG diluted with 1% Skimmed Milk (in PBS, pH 7.2) for 1 hour at room temperature.
10) Wash the membrane with PBS (5 minutes x 6 times).
11) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap.
12) Expose to an X-ray film in a dark room for 3 minutes. Develop the film as usual. The conditions for exposure and development may vary.
Positive control for Western blotting; transfectant

Flow cytometric analysis for floating cells
Protocol 1
We usually use Fisher tubes or equivalents as reaction tubes for all steps described below.
1) Wash the cells 3 times with washing buffer [PBS containing 2% fetal calf serum (FCS) and 0.1% NaN3].
2) Resuspend the cells with washing buffer (5x10e6 cells/mL).
3) Add 50 µL of the cell suspension into each tube, and centrifuge at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
4) Add 10 µL of normal goat serum containing 1 mg/mL normal human IgG and 0.1% NaN3 to the cell pellet after tapping. Mix well and incubate for 5 minutes at room temperature (20~25 oC).
5) Add 30 µL of the anti-human BAFF-R/BR3 monoclonal antibody (8A7) (5 µg/mL) diluted with the washing buffer. Mix well and incubate for 30 minutes at room temperature (20~25 oC).
6) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
7) Add 30 µL of 1:40 FITC conjugated anti-mouse IgG diluted with the washing buffer. Mix well and incubate for 15 minutes at room temperature (20~25oC).
8) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
9) Resuspend the cells with 500 µL of the washing buffer and analyze by a flow cytometer.
Positive control for flow cytometry; transfectant

Protocol 2
We usually use Fisher tubes or equivalents as reaction tubes for all steps described below.
1) Wash the cells 3 times with washing buffer [PBS containing 2% fetal calf serum (FCS) and 0.1% NaN3].
2) Resuspend the cells with PBS containing 25% normal goat serum, 1 mg/mL normal human IgG and 0.1% NaN3 (5x10e6 cells/mL).
3) Add 30 µL of the anti-human BAFF-R/BR3 monoclonal antibody (8A7) (50 µg/mL) diluted with the washing buffer into each tube.
4) Add 50 µL of the cell suspension into each tube. Mix well and incubate for 30 minutes at room temperature (20~25 oC).
5) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
6) Resuspend the cells with 50 µL of the washing buffer.
7) Add 30 µL of 1:40 FITC conjugated anti-mouse IgG diluted with the washing buffer into each tube. Mix well and incubate for 15 minutes at room temperature (20~25oC).
8) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
9) Resuspend the cells with 500 µL of the washing buffer and analyze by a flow cytometer.
Positive control for flow cytometry; transfectant

Flow cytometric analysis for whole blood cells
We usually use Falcon tubes or equivalents as reaction tubes for all steps described below.
1) Add 20 µL of the anti-human BAFF-R/BR3 monoclonal antibody (8A7) (5 µg/mL) diluted with the washing buffer [PBS containing 2% fetal calf serum (FCS) and 0.1% NaN3] into each tube.
2) Add 50 µL of whole blood into each tube. Mix well, and incubate for 30 minutes at room temperature (20~25 oC).
3) Add 1 mL of washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
4) Add 30 µL of 1:40 FITC conjugated anti-mouse IgG diluted with washing buffer. Mix well and incubate for 15 minutes at room temperature (20~25oC).
5) Add 1 mL of washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
6) Lyse with OptiLyse C (for analysis on Beckman Coulter instruments) or OptiLyse B (for analysis on BD instruments), using the procedure recommended in the respective package inserts.
7) Add 1ml of H2O to each tube and incubate for 10 minutes at room temperature (20~25 oC).
8) Centrifuge at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
9) Add 1 mL of washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
10) Resuspend the cells with 500 µL of the washing buffer and analyze by a flow cytometer.

Immunohistochemical staining for paraffin embedded sections: SAB method
1) Deparaffinize the sections with Xylene 3 times for 3-5 minutes each.
2) Wash the slides with Ethanol 3 times for 3-5 minutes each.
3) Wash the slides with PBS 3 times for 3-5 minutes each.
4) Heat treatment Heat treatment by microwave oven: Place the slides put on staining basket in 500 mL beaker with 500 mL citrate buffer (pH 6.5). Cover the beaker with plastic wrap, then process the slides 2 times for 10 minutes each at 500 W with microwave oven. Let the slides cool down in the beaker at room temperature for about 40 minutes.
5) Remove the slides from the citrate buffer and cover each section with 3% H2O2 for 10 minutes at room temperature to block endogenous peroxidase activity. Wash 3 times in PBS for 5 minutes each.
6) Remove the slides from PBS, wipe gently around each section and cover tissues with Protein Blocking Agent for 5 minutes to block non-specific antibody staining. Do not wash.
7) Tip off the blocking buffer, wipe gently around each section and cover tissues with the anti-human BAFF-R/BR3 monoclonal antibody (8A7) diluted with PBS containing 1% BSA as suggested in the APPLICATIONS.
8) Incubate the sections for 1 hour at room temperature.
9) Wash the slides 3 times in PBS for 5 minutes each.
10) Wipe gently around each section and cover tissues with Polyvalent Biotinylated Antibody. Incubate for 10 minutes at room temperature. Wash as in step 9).
11) Wipe gently around each section and cover tissues with Streptavidin-Peroxidase. Incubate for 10 minutes at room temperature. Wash as in step 9).
12) Visualize by reacting for 10-20 minutes with substrate solution containing 7.5 mg DAB, 40 µL of 30% H2O2 in 150 mL PBS.
*DAB is a suspected carcinogen and must be handled with care. Always wear gloves.
13) Wash the slides in water for 5 minutes.
14) Counter stain in hematoxylin for 1 minute, wash the slides 3 times in water for 5 minutes each, and then immerse the slides in PBS for 5 minutes. Dehydrate by immersing in Ethanol 3 times for 3 minutes each, followed by immersing in Xylene 3 times for 3 minutes each.
15) Now ready for mounting.
Positive control for Immunohistochemistry; transfectant

General Readings
  1. Mackay, F., et al., Annu. Rev. Immunol. 21, 231-264 (2004).
Storage Upon receipt, store (in aliqouts) at -20 °C. Avoid repeated freezing and thawing.
Shelf life: One year from despatch.
Format
Purification:
Protein A agarose
Buffer System:
PBS containing 50% glycerol, pH 7.2. Contains no preservatives.
State:
Liquid Ig fraction
Azide Free
Specificity
Specificity:

This antibody reacts with human BAFF-R/BR3.

Gene ID 115650

Accessory Products

Proteins and/or Positive Controls

Proteins for CD268 / BAFFR (9 products)

Catalog No. Species Pres. Purity   Source  

CD268 / BAFFR

CD268 / BAFFR Human > 95 %
Preparation: .
Purity Detail: >95% as determined by SDS-PAGE and Coomassie blue staining.
E. coli

Regular Price: 50 µg / €199.00

Special Price: 50 µg / €139.00

  OriGene Technologies, Inc.

CD268 / BAFFR

CD268 / BAFFR Human Purified > 95 % pure by SDS-PAGE gel and HPLC analyses E. coli
  Acris Antibodies GmbH

CD268 / BAFFR

CD268 / BAFFR Human Purified > 95 % pure by SDS-PAGE gel and HPLC analyses E. coli
  Acris Antibodies GmbH

CD268 / BAFFR

CD268 / BAFFR Human
  Abnova Taiwan Corp.

CD268 / BAFFR

CD268 / BAFFR Human Purified 12.5% SDS-PAGE Stained with Coomassie Blue.
  Abnova Taiwan Corp.

CD268 / BAFFR

CD268 / BAFFR Human Purified 12.5% SDS-PAGE Stained with Coomassie Blue.
  Abnova Taiwan Corp.

CD268 / BAFFR

CD268 / BAFFR Human
  Abnova Taiwan Corp.

CD268 / BAFFR

CD268 / BAFFR Human Purified
  Novus Biologicals Inc.

CD268 / BAFFR

CD268 / BAFFR Human Purified
  Novus Biologicals Inc.

Positive controls for CD268 / BAFFR (2 products)

Catalog No. Species Pres. Purity   Source  

BAFF Receptor Lysate

Western Blot: BAFF Receptor Lysate [NBL1-17149] - Western Blot experiments. 
Left-Control; Right -Over-expression Lysate for TNFRSF13C
  Novus Biologicals Inc.

TNFRSF13C 293T Cell Transient Overexpression Lysate(Denatured)

TNFRSF13C 293T Cell Transient Overexpression Lysate(Denatured)
  Abnova Taiwan Corp.
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