NewBlot Nitro Stripping Buffer for Westerns on Nitrocellulose.

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.

Choosing the Right Stripping Buffer

Specifications

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.

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.

Amount of time blot is in stripping buffer

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.

Blot handling conditions

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.

NewBlot™ Nitro Stripping Buffer for Nitrocellulose Membranes