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IRDye® 680LT Protein Labeling Kit - High Molecular Weight

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Labeling (Lbl)
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原理 The labeling kit contains IRDye 680LT activated with NHS ester reactive group that will couple to free amino groups and form a stable conjugate with high molecular weight proteins (45 - 200 kDa).
样品类型 Variable (Protein containing Samples)
检测方法 Fluorometric
特异性 Western blot applications
产品特性 Labeled proteins may be used for Western blots and other applications. Fluorescent conjugates labeled with IRDye 680LT display an absorption maximum of 680 nm and an emission maximum of 694 nm in 1X PBS. These spectral characteristics match the 700 nm channel of Odyssey® Imaging Systems.
The IRDye 680LT dye bears an NHS ester reactive group that will couple to free amino groups and form a stable conjugate with high molecular weight proteins (45 - 200 kDa). Fluorescent conjugates labeled with IRDye680LT display an absorption maximum of 680 nm and an emission maximum of 694 nm in 1X PBS.
组件 3 x IRDye 680LT Reactive Dye vials (0.1 mg) (store at -20 °C) 1 x 0.5 mL 1 M Potassium Phosphate (K2HPO4), pH 9 (store at 4 °C) 1 x 25 mL 1X PBS (store at 4 °C) 1 x 0.5 mL ultra pure water (store at 4° C) 3 x Pierce® Zeba™ Desalting Spin Columns, P/N 89891 (store at 4 °C) Note: The minimum recommended protein molecular weight for these columns is 7 kDa. Pierce Zeba Desalting Spin Column instructions Protocol for IRDye 680LT Protein Labeling Kit - High MW
应用备注 Optimal working dilution should be determined by the investigator.
实验时间 2 - 3 h
实验流程 As an example protocol we would mention the following:
I. Required Reagents

Blotted nitrocellulose (LI-COR, P/N 926-31090/926-31092) or Immobilon®-FL PVDF mem-brane (LI-COR, P/N 926-31099 or 926-31100)
Odyssey® Blocking Buffer (PBS) (LI-COR, P/N 927-40000, 927-40100) or Odyssey Blocking Buffer (TBS) (LI-COR, P/N 927-50000, 927-50100)**
Primary antibodies
IRDye® 800CW, 680RD, or 680LT secondary antibodies (LI-COR)
Tween® 20
Ultrapure water
Methanol for wetting of PVDF
** IMPORTANT! PBS-based and TBS-based blocking buffers may be used with this protocol. Odyssey Blocking Buffer is available in both formulations. Be sure to keep your buffer system consistent throughout the protocol for blocking, antibody dilutions, and washes. For example, if you use a TBS-based buffer system, choose Odyssey Blocking Buffer (TBS). If you use a PBS-based buffer system, choose Odyssey Blocking Buffer (PBS).

As an examle protocol we would mention the following:
II. Quick Start Hints and Tips

Infrared fluorescence detection with Odyssey Family Imaging Systems provides a quantitativetwo-color detection method for Western blots. Following, you will find some basic Hints and Tipsto help you get started.
1. Store the IRDye secondary antibody vial in darkness at 4 °C. Minimize exposure to light andtake care not to introduce contamination into the vial. Dilute immediately prior to use. If particulates are seen in the antibody solution, centrifuge before use.
Page 2 - Near-Infrared (NIR) Western Blot Detection
2. The best transfer conditions, membrane, and blocking agent for experiments will vary, de-pending on the antigen and antibody.
3. Do not write on blot with a pen or Sharpie® marker. Ink from most pens and markers will fluoresce in the 700 nm channel of all Odyssey® Family Imaging Systems. The ink may wash off and re-deposit elsewhere on the membrane, causing increased background. Use a pencil to mark membranes. Ink from the Odyssey Pen does not fluoresce and can be used to mark nitrocellulose membranes. If the Odyssey Pen is used for PVDF membranes, the ink will dissolve and wash off when the blot is wetted in methanol.
4. Let the membrane dry after transfer for 1 hour or overnight, to maximize protein retention on the membrane.
5. Handle blot with clean forceps only.
6. Before using forceps, incubation trays, and the Odyssey scanning surface or sample tray (if applicable), clean with 100 % methanol to remove any residual dye signal from previous use. Rinse with a small volume of distilled water, followed by isopropanol. Dry with a lint-free wipe.
7. When processing Western blots, do not use dishes/boxes that have ever been used for Coomassie staining. The Odyssey imagers are very sensitive to Coomassie (which is a strongly-fluorescent dye), and use of dishes with small traces of Coomassie will add a tremendous amount of background in the 700 nm channel.
Maintain the same buffer system throughout the Western blot process. For example, if youblock your blot in Odyssey Blocking Buffer (PBS), use PBS-based buffers throughout the protocol.
8. Do not include detergents during the blocking step.
9. For 1-color blots, use IRDye® 800CW secondary antibody for detection of the protein for best sensitivity.
10. For 2-color blots:
Make sure primary antibodies are from different species (for example, Rabbit and Mouse)
Use the IRDye 800CW secondary antibody to detect the protein target with lowest abundance, and IRDye 680RD secondary antibody to detect the more abundant protein.
11. If you are using PVDF, add 0.01 % SDS to the diluted secondary antibody. Do not add SDS ifusing nitrocellulose membrane.
12. Incubate with secondary antibodies in the dark for one hour with gentle shaking. The incu-bation box can be covered with aluminum foil.
Near-Infrared (NIR) Western Blot Detection

III. Western Blot Detection Methods

This protocol is designed to help you achieve success with NIR Western blot detection methods.Read the entire protocol carefully before beginning your optimization experiments.
Membrane Guidelines
A low-background membrane is essential for NIR Western blot success. Background can resultfrom membrane autofluorescence or from non-specific binding of antibodies. Polyvinylidene fluo-ride (PVDF) and nitrocellulose membranes are typically used for Western blotting applications.There are many brands and vendors for both types of membrane.
Before using your blotting membrane for the full Odyssey® Western blot protocol, cut a smallsample of membrane for testing. Image this sample (both wet and dry) to evaluate the level ofmembrane autofluorescence. If possible, include a sample of membrane that is known to workwell with the Odyssey system, so you can compare background levels.
To learn more about optimizing your Western blots, see Good Westerns Gone Bad: Tips to MakeYour NIR Western Blot Great (
Western Blot Detection Protocol
After membrane transfer, perform the following steps:
1. For PVDF membrane:
a. Pre-wet 1 minute in 100 % methanol.
b. Rinse with ultra pure water.
c. Wet in 1X PBS or TBS for 2 minutes (using the appropriate buffer system).
For nitrocellulose membrane:
a. Wet in 1X PBS or TBS for 2 minutes, or until fully hydrated (using the appropriate buffer system).
2. Place membrane in incubation box and block the membrane in Odyssey Blocking Buffer (PBS or TBS) for 1 hour with gentle shaking. Be sure to use sufficient blocking buffer to cover the membrane (a minimum of 0.4 mL/cm2 is suggested).
3. Prepare primary antibody:
a. Primary antibody diluent: Odyssey Blocking Buffer (PBS or TBS) +0.2 % Tween® 20 (final concentration).
b. Dilute primary antibody in Odyssey Blocking Buffer + 0.2 % Tween 20, using the ven-dor's recommended dilution for Western blot applications. Dilutions typically range from 1:200 - 1:5,000, depending on the primary antibody.
c. Use enough antibody solution to completely cover the membrane.
4. Incubate blot in diluted primary antibody for 1 to 4 hours at room temperature with gentle shaking, or overnight at 4 °C.
Optimal incubation times vary for different primary antibodies.
If the procedure cannot be completed in full, this is a good place to stop until the following day. Incubate the blot in primary antibody overnight at 4 °C with gentle shaking, and re-sume the protocol the next day.
5. Wash membranes:
• Pour off primary antibody solution.
• Rinse membrane with appropriate buffer - 1X TBS-T (0.1 % Tween® 20) or 1X PBS-T (0.1 % Tween 20).
• Cover blot with 1X TBS-T (0.1 % Tween 20) or 1X PBS-T (0.1 % Tween 20).
• Shake vigorously on platform shaker at room temperature for 5 minutes.
• Pour off wash solution.
• Repeat 3 additional times.
6. Dilute IRDye® secondary antibody in the appropriate diluent listed below:
a. Secondary antibody diluent: PVDF membrane
• To blocking buffer, add Tween 20 to a final concentration of 0.2 % and SDS to a final concentration of 0.01 %.
b. Secondary antibody diluent: nitrocellulose membrane
• To blocking buffer, add Tween 20 to a final concentration of 0.2 %. Do not add SDS.
NOTE: A Suggested dilution range for secondary antibodies is typically 1:5,000 to 1:25,000. Recommended dilutions can be found on the secondary antibody pack insert. Use 1:20,000 as a suggested starting point if using LI-COR secondary antibodies.
7. Protect membrane from light during incubation. Incubate blot in diluted secondary antibody for one hour at room temperature with gentle shaking.
8. Protect membrane from light during washes.
Wash membranes:
• Pour off secondary antibody solution.
• Rinse membrane with the appropriate buffer - 1X TBS-T (0.1 % Tween 20) or 1X PBS-T (0.1 % Tween 20).
• Cover blot with 1X TBS-T (0.1 % Tween 20) or 1X PBS-T (0.1 % Tween 20).
• Shake vigorously on platform shaker at room temperature for 5 minutes.
• Pour off wash solution.
• Repeat 3 additional times.
9. Rinse membrane with 1X TBS/PBS (as appropriate) to remove residual Tween 20.
10. The Western blot is now ready to image.
• The membrane can be stored in 1X TBS or 1X PBS for up to 48 hours in the dark at 4 °C.
If the blot is prepared more than 48 hours in advance, air-dry the blot and store in the dark at room temperature until ready to image.
11. Image with an Odyssey® Family Imaging System.
• If signal on membrane is too strong or too weak, adjust the imaging parameters to opti-mize the image.
- Odyssey Classic: Re-image the membrane at a lower or higher scan intensity setting, respectively.
- Odyssey Fc: Adjust image acquisition time.
- Odyssey CLx: Use the AutoScan function to improve the dynamic range of the image.

IV. Guidelines for Two-Color Detection

Two different antigens can be detected simultaneously on the same blot using IRDye® secondaryantibodies. When performing a two-color blot, use the standard Western blot protocol with thefollowing modifications:
• Combine the two primary antibodies in the diluted antibody solution (Step 3, Section III). Incubate simultaneously with membrane (Step 4, Section III). The primary antibodies must be from two different host species.
• Combine the two IRDye® secondary antibodies in the diluted antibody solution (Step 6, Section III). Incubate simultaneously with membrane (Step 7, Section III).
Two-color detection requires careful selection of primary and secondary antibodies. The followingguidelines will help you successfully design two-color experiments:
• The two primary antibodies must be derived from different host species so that they can bediscriminated by secondary antibodies of different specificities (for example, primary anti-bodies from rabbit and mouse will be discriminated by anti-rabbit IgG and anti-mouse IgG secondary antibodies, respectively).
• If the two primary antibodies are mouse monoclonals from different IgG subclasses (IgG1, IgG2a, or IgG2b), IRDye Subclass-Specific secondary antibodies can be used for multiplex detection. The same subclasses cannot be combined in a two-color Western blot (for exam-ple, two IgG1 primary antibodies). For details, refer to Western Blot and In-Cell Western™ Assay Detection Using IRDye Subclass-Specific Antibodies
• Anti-Goat secondary antibodies cannot be multiplexed with Goat-derived secondary anti-bodies (Example: Donkey anti-Goat and Goat anti-Rabbit). The secondary antibodies will cross-react.
• One secondary antibody must be labeled with a 700 nm channel dye and the other with 800 nm channel dye.
• In general, it is recommended that the IRDye® 800CW secondary antibody (800 nm channel) be used to detect the lower-abundance protein target and IRDye 680RD sec-ondary antibody (700 nm) to detect the more abundant protein.

• Always use highly cross-adsorbed secondary antibodies for two-color detection. Failure to use cross-adsorbed antibodies may result in increased cross-reactivity of the second-ary antibodies.
• For best results, avoid using primary antibodies from mouse and rat together in a two-color experiment. The two species are so closely related that it is not possible to com-pletely adsorb away all cross-reactivity. If there is no other option, it is crucial to run sin-gle-color blots first with each individual antibody to be certain of expected band sizes.
V. Adapting Western Blot Protocols for Odyssey® Imaging Systems When adapting Western blotting protocols for Odyssey detection or using a new primary anti-body, it is important to determine the optimal antibody concentrations. Optimization will helpachieve maximum sensitivity and consistency, while minimizing background. Three parametersshould be optimized: primary antibody concentration, IRDye secondary antibody concentration,and detergent concentration in the diluted antibodies.
Primary Antibody Concentration
Primary antibodies vary widely in quality, affinity, and concentration. The correct working rangefor antibody dilution depends on the characteristics of the primary antibody and the amount oftarget antigen to detect. Start with the vendor's dilution recommendation for Western blotting orwith the dilution normally used for chemiluminescent detection.
Secondary Antibody Concentration
Optimal dilutions of IRDye secondary antibodies should also be determined. Refer to the appro-priate pack insert for recommendations at The amount of sec-ondary antibody required varies depending on how much antigen is being detected. Abundantproteins with strong signals may require less secondary antibody. Using too much secondary antibody may increase membrane background and/or non-specific banding.
Detergent Concentration
Addition of detergents to diluted antibodies can significantly reduce background on the blot. Optimal detergent concentration will vary, depending on the antibodies, membrane type, andblocker used. Keep in mind that some primary antibodies do not bind as tightly as others and maybe washed away by too much detergent. Never expose the membrane to detergent until blockingis complete, as this may cause high membrane background.
Tween® 20:
• Blocking buffer - do not add Tween 20 during blocking.
• Diluted primary and secondary antibodies should contain Tween 20.
Use a final concentration of 0.1 - 0.2 % Tween 20 for PVDF or nitrocellulose membranes.
• Wash solutions should contain 0.1 % Tween 20.

When using PVDF membrane, addition of SDS will dramatically reduce overall mem-brane background in the 700 nm channel. It is critical to use only a very small amount, because SDS is an ionic detergent and can disrupt antibody-antigen interactions if too much is present at any time during the protocol. SDS should not be used with nitrocellulose membranes.
• Blocking buffer - do not add SDS to the blocking reagent during blocking.
• Diluted primary antibodies should not contain SDS.
• Diluted secondary antibodies should contain a final concentration of 0.01 - 0.02 % SDS and 0.1 - 0.2 % Tween® 20, when PVDF membrane and IRDye® 680LT secondary antibodies are used.
• SDS is optional when using IRDye 680RD antibodies with PVDF membrane, but is essential when using IRDye 680LT antibodies with PVDF.
• Wash solutions should not contain SDS.

限制 仅限研究用
储存液 Sodium azide
注意事项 This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
储存条件 -20 °C,4 °C
储存方法 Dye vials (-20°C), all other components at 4°C