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.
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.