Assays that use IRDye 800CW conjugates (such as in vivo imaging and cell binding assays) may require a “dye-only” control for potential effects or retention of the dye.
Carboxylate (non-reactive) form of IRDye 800CW is an ideal control.
Note: The carboxylate dye has no reactive group and cannot be used for labeling.
Clearance kinetics of IRDye 800CW carboxylate. A single SCID mouse was injected with 1 nmol of non-reactive IRDye 800CW dye, and clearance monitored over time as indicated. Pseudocolor fluorescence is superimposed on a white light image. IRDye 800CW carboxylate dispersed rapidly and was completely cleared after 48 h. Originally published in Kovar, J., et al. (2007) Anal Biochem 367:1-12.IRDye 800CW 羧酸盐的清除动力学。 给一只 SCID 小鼠注射 1 nmol 的非反应性 IRDye 800CW 染料，并如图所示随时间监测清除率。 伪彩色荧光叠加在白光图像上。 IRDye 800CW 羧酸盐迅速分散，48 小时后完全清除。 最初发表于 Kovar, J. 等人。 (2007) 肛门生物化学 367:1-12。
How is IRDye 800CW Carboxylate Used?
In vitro Cell-based Assays
As a control for cell-based assays that monitor binding of a dye-labeled agent.
Validation of optical agents for in vivo administration
Evaluation of binding specificity
In vivo Imaging
For evaluation of behavior and clearance of the dye itself.
Timing of dye clearance from the animal’s body
Retention of dye in certain organs or sites (e.g., liver or kidneys)
Labeling Reaction Reference
As a standard to determine the amount of unreacted (“free”) dye after IRDye 800CW conjugation and purification.
Residual unreacted dye may cause:
Artificially high values when dye/protein (D/P) ratio is calculated
Increased background fluorescence in biological assays
NewBlot Nitro is optimized for near-infrared (NIR) Western blots and is specially formulated for use with IRDye® secondary antibodies, including IRDye 800CW, IRDye 680RD, and IRDye 680LT.
NewBlot Nitro is intended for nitrocellulose membranes only, and is ideal for removal of secondary antibody.
NewBlot Nitro Stripping Buffer is supplied as a 5X concentrated solution. Each 100 mL bottle is sufficient for up to 3000 cm2 or approximately fifty 7 x 8.5 cm Odyssey® nitrocellulose membranes.
NewBlot Nitro stripping buffer contains hazardous materials and additional shipping costs will be applied.
NewBlot Nitro Stripping Buffer 以 5X 浓缩溶液的形式提供。 每个 100 mL 瓶足以容纳 3000 cm2 或大约 50 个 7 x 8.5 cm Odyssey® 硝酸纤维素膜。
NewBlot Nitro 剥离缓冲液含有有害物质，将收取额外的运输费用。
Example Data
Example of stripping and reprobing with NewBlot Nitro Stripping Buffer. Rabbit anti-β-tubulin and mouse anti-ERK2 were run on a nitrocellulose membrane Western blot. The blot was probed with IRDye 680 Goat anti-Rabbit (red) and IRDye 800CW Goat anti-Mouse (green). The blot was stripped with NewBlot Nitro Stripping Buffer (1X) and reprobed three sequential times with the same antibodies. All blots have been scanned at 169 micron resolution and scan intensity of 6 on the Odyssey Infrared Imaging System.
Factors Affecting Efficiency when Using NewBlot Nitro Stripping Buffer
Recommended Standard Conditions: 1X NewBlot Nitro, 5 minutes at room temperature.
Below are key factors that affect stripping efficiency with NewBlot Nitro on Odyssey nitrocellulose membranes. The data figure above compares stripping efficiency using various concentrations, time, and temperature to the recommended standard conditions, 1X, 5 min, ambient.
For optimal stripping results, follow the optimization guidelines in NewBlot Nitro Stripping Buffer pack insert.
Increasing stripping time has the greatest effect on efficiency.
Increasing the stripping time may lead to increased damage/loss of target antigens, and reduce the success of reprobing.
Sample type and preparation
Even under the most stringent stripping conditions, the fluorescent signal may not be removed completely due to sample load amount, antibody affinity/avidity, and target protein abundance.
Washing, scanning, or stripping efficiency will be affected if the blot is allowed to dry at all during incubation. Keep the blot moist at all times.
Buffer concentration and temperature used for stripping
Increasing the stripping buffer concentration and temperature significantly improves stripping effectiveness but can also have a highly detrimental effect on reprobing.
If the optimization process given in the protocol does not produce the desired stripping results, stripping buffer incubation can be carried out at 37 °C using a water bath or warm-air incubator.
Do not microwave the NewBlot Nitro Stripping Buffer or the nitrocellulose blot.
