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5151
Anti-rabbit IgG (H+L) (DyLight™ 800 4X PEG Conjugate)
Secondary Antibodies
Secondary Antibody

Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) #5151

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  1. WB
Western Blotting Image 1: Anti-rabbit IgG (H+L) (DyLight™ 800 4X PEG Conjugate)
Western blot analysis of Jurkat cell lysates (#9194) treated with either U0126 (MEK 1/2 inhibitor) #9903 or TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/204) (D13.14.4E) XP® Rabbit mAb #4370 detected with Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) (green) and p44/42 MAPK (Erk1/2) (3A7) Mouse mAb #9107 detected with Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470 (red). The array image pixel intensities obtained using a LI-COR® Biosciences Odyssey® Infrared Imaging System are shown in the upper panel while corresponding fluorescent western blots are shown in the lower panel.
Product Image 1: Anti-rabbit IgG (H+L) (DyLight™ 800 4X PEG Conjugate)
In-Cell Western analysis of A549 cells exposed to varying concentrations of U0126 (MEK1/2 Inhibitor) #9903 for 3 hours, followed by TPA (Phorbol-12-Myristate-13-Acetate) #9905 stimulation for 30 minutes. With increasing concentrations of U0126, a significant decrease (~5 fold) in Phospho p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 signal as compared to the TPA-stimulated control was observed. When using phospho-Erk as a measurement, the IC50 of this compound was 2.8 μM. Data and images were generated on the LI-COR® Biosciences Odyssey® Infrared Imaging System using Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate). DRAQ5® #4084 (fluorescent DNA dye - red) is used for normalization.
To Purchase # 5151
Cat. # Size Qty. Price
5151P
100 µl
$ 88
5151S
500 µl
$ 180

Supporting Data

REACTIVITY
SENSITIVITY
MW (kDa)
Source/Isotype Goat 

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
  • All-All Species Expected

Product Description

Anti-rabbit IgG (H+L) was conjugated to DyLight™ 800 4X PEG fluorescent dye under optimal conditions and formulated at 1 mg/ml. Excitation is 777 nm and peak fluorescence emission is 794 nm.

Product Usage Information

The optimal dilution of the anti-species antibody should be determined by the user. However, the final dilutions below should yield acceptable results for the respective applications.

Fluorescent western blotting: 1:30000

In-Cell Western: 1:1000

Storage

Proprietary buffer. Store at 4°C. Protect from Light. Do not freeze.

Protocol

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Western Blotting Protocol (Fluorescent)

NOTE: The TrueBlack® Fluorescent Western Blot Blocking Buffer Kit (#40683) contains the necessary buffers to block the membrane and dilute the primary and secondary antibodies.

NOTE: Two-color western blots require primary antibodies from different species and appropriate secondary antibodies labeled with different dyes. Overlap of epitopes may cause interference and should be considered in two color western blots. If the primary antibodies require different primary antibody incubation buffers, test each primary individually in both buffers to determine the optimal one for the dual-labeling experiment.

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 TBS 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 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST-10X): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Wash Buffer: 1X TBST.
  8. TrueBlack® WB Blocking Buffer: (#57443) Ready-to-use solution.
  9. TrueBlack® WB Antibody Diluent: (#78710) Ready-to-use solution. (Secondary antibodies; anti-rabbit #5151 and #5366; anti-mouse #5257 and #5470).
  10. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  11. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes (recommended). Pore size 0.2 µm is generally recommended.

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 cold 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl per plate of 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) is recommended to verify electrotransfer and to determine molecular weights. Prestained markers are autofluorescent at near-infrared wavelengths.

  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.

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Warm the TrueBlack® WB Blocking Buffer to room temperature and thoroughly mix prior to use.

    NOTE: The TrueBlack® WB Blocking Buffer may have a slight precipitate but this will not impact the performance.

  3. Incubate membrane in 10 ml of TrueBlack® WB Blocking Buffer for 45 min at room temperature.
  4. Remove the TrueBlack® WB Blocking Buffer.
  5. Incubate membrane and primary antibody (at the appropriate dilution as recommended in the product datasheet) in 10 ml of TrueBlack® WB Antibody Diluent with gentle agitation overnight at 4°C.
  6. Wash five times for 10 min each with 15 ml of TBST.
  7. Incubate membrane with fluorophore-conjugated secondary antibody (#5470, #5257, #5366, #5151) (1:5000–1:25,000 dilution of 1 mg/ml stock) in 10 ml of TrueBlack® WB Antibody Diluent with gentle agitation for 2 hr at room temperature, protect from light.
  8. Wash five times for 10 min each with 15 ml of TBST, protect from light.

D. Detection of Proteins

  1. Drain membrane of excess TBST and allow to dry.

    CRITICAL STEP: Membrane must be dry for fluorescent staining.

  2. Scan membrane using an appropriate fluorescent scanner following the manufacturer’s recommendations.

Protocol Id: 2544

Protocol Id:

Specificity / Sensitivity

Anti-rabbit IgG (H+L) (DyLight™ 800 4X PEG Conjugate) reacts with heavy and light chain of most rabbit immunoglobulins. No cross-reactivity to other serum proteins has been detected. This antibody may cross-react with immunoglobulins from other species.

Source / Purification

This antibody is prepared from goat antibodies and purified by immunoaffinity chromatography using antigen coupled to agarose beads.

Background

Near infrared anti-species IgG conjugates are ideal for fluorescent western blotting and In-Cell Western. Cell Signaling Technology's strict quality control procedures assure that each conjugate provides optimal specificity and fluorescence.
This product has been optimized for use as a secondary antibody in fluorescent western blotting and In-Cell Western™.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
LI-COR is a registered trademark of LI-COR, Inc.
Odyssey is a registered trademark of LI-COR, Inc.