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5571
Anti-biotin (D5A7) Rabbit mAb (HRP Conjugate)
Secondary Antibodies
Secondary Antibody

Anti-biotin (D5A7) Rabbit mAb (HRP Conjugate) #5571

Citations (27)
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  1. WB
Western blot analysis of extracts from HeLa cells, untreated or treated with Staurosporine #9953 (1 μM, 3 hr) using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb (Biotinylated) primary antibody and Anti-Biotin (D5A7) Rabbit mAb (HRP Conjugate) as secondary detection antibody.
Relative fluorescence of biotinylated and non-biotinylated peptides determined by #5571 Anti-biotin (D5A7) Rabbit mAb (HRP Conjugate) detection using #7003 LumiGLO® Reagent and 20X Peroxide reagents.
Chemiluminescent detection of phosphorylated and non-phosphorylated Met peptides with #4033 Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb (Biotinylated) in conjunction with #5571 Anti-biotin (D5A7) Rabbit mAb (HRP Conjugate).
To Purchase # 5571
Cat. # Size Qty. Price Inventory
5571S
1 ml

Supporting Data

REACTIVITY Rab
SENSITIVITY
MW (kDa)
Source/Isotype Rabbit 

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Description

Anti-biotin (D5A7) Rabbit mAb is conjugated to the carbohydrate groups of horseradish peroxidase (HRP) via its amine groups. This product has been optimized to detect biotinylated primary antibodies. HRP conjugated antibodies do not require incubation with a secondary antibody. *Do not mix this antibody in solution with any Anti-rabbit antibody. Anti-rabbit antibodies will cross react with this antibody and could result in decreased activity of both Anti-rabbit and Anti-biotin (D5A7) Rabbit mAb.

Product Usage Information

Recommended Antibody Dilutions:

ELISA-peptide 1:1000

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 2 mg/ml bovine serum albumin (BSA), and 50% glycerol. Store at –20°C. Do not aliquot the antibody.

Protocol

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
  11. Biotinylated Protein Ladder Detection Pack: (#7727).
  12. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  13. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  14. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

For HRP Conjugated Primary Antibodies

  1. Incubate membrane and primary antibody (at the appropriate dilution as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate with Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000), to detect biotinylated protein markers, in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 11

ELISA-Peptide Protocol for HRP Conjugates

A. Solutions and Reagents

  1. Carbonate Buffer: 15 mM Na2CO3, 35 mM NaHCO3, 0.2 g/l NaN3 (pH 9.6). Use 1 µM synthetic peptide in carbonate buffer.
  2. 10X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 l add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 l dH2O. Adjust pH to 7.4.
  3. Wash Buffer: 1X PBS containing 0.05% Tween-20 (PBST) (9809)
  4. Blocking Buffer: 10 mg/ml bovine serum albumin (BSA) (9998) in PBST
  5. Primary Antibody Dilution Buffer: 1 mg/ml BSA in PBST
  6. Secondary Antibody Dilution Buffer: 3% BSA in PBST
  7. 96-Well Plate: Solid white or opaque plates are recommended for chemiluminescent detection.

B. Binding Peptides to 96-Well Plate

  1. Coat the wells of a 96-well microtiter plate with 100 µl of 1 µM synthetic peptide in carbonate buffer by incubating overnight at 4°C or for 2 to 6 hours at 37°C. If the peptide does not bind or absorb, try other buffers in the pH 4–8 range.
  2. Wash plate three times 200 µl/well with wash buffer.
  3. Block plate with 200 µl/well blocking buffer for 1 hour at 37°C. Wash plate three times with wash buffer. (May leave dry plate at 4°C for 1–2 months if desired.)

C. Protocol for HRP-Conjugated Primary Antibody

  1. Prepare recommended dilution of HRP-conjugated primary antibody with primary antibody dilution buffer. Add 100 µl to wells and incubate at 37°C for 1 hour.
  2. Wash five times with wash buffer.
  3. Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution (#7003) and Stable Peroxide Buffer.
  4. Use a plate-based luminometer to measure Relative Light Units (RLU) at 425 nM within 1–10 minutes following addition of the substrate.
  5. Optimal signal intensity is achieved when read within 10 minutes.

D. Protocol for HRP-Conjugated Secondary Antibody

  1. Prepare recommended dilution of primary antibody with primary antibody dilution buffer. Add 100 µl to wells and incubate overnight at 4°C or 2 to 6 hours at 37°C.
  2. Wash three times with wash buffer.
  3. Prepare recommended dilution of HRP-conjugated Anti-mouse IgG, HRP-linked Antibody #7076 with secondary antibody dilution buffer. Add 100 µl to wells and incubate at 37°C for 1 hour.
  4. Wash five times with wash buffer.
  5. Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution (# 7003) and Stable Peroxide Buffer.
  6. Use a plate-based luminometer to measure Relative Light Units (RLU) at 425nM within 1–10 minutes following addition of the substrate.
  7. Optimal signal intensity is achieved when read within 10 minutes.

posted October 2010

protocol id: 1

Protocol Id: 1

Specificity / Sensitivity

Anti-biotin (D5A7) Rabbit mAb (HRP Conjugate) recognizes biotin attached to proteins, peptides, oligonucleotides and solid matrices.

Species Reactivity:

Rabbit

Source / Purification

Monoclonal antibody is produced by immunizing animals with a biotinylated protein.

Background

Chemiluminescence systems have emerged as the best all-around method for western blot detection. They eliminate the hazards associated with radioactive materials and toxic chromogenic substrates. The speed and sensitivity of these methods are unequalled by traditional alternatives, and because results are generated on film, it is possible to record and store data permanently. Blots detected with chemiluminescent methods are easily stripped for subsequent reprobing with additional antibodies. HRP-conjugated secondary antibodies are utilized in conjunction with specific chemiluminescent substrates to generate the light signal. HRP conjugates have a very high turnover rate, yielding good sensitivity with short reaction times.

Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
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