NewBlot IR Stripping Buffers for NIR Western Blots

NewBlot IR Stripping Buffer for PVDF or Nitrocellulose Membranes.

NewBlot IR is a robust formulation optimized for near-infrared (NIR) Western blots that works with both nitrocellulose and PVDF membranes. This stripping buffer is a good place to start if you are new to stripping and reprobing NIR Western blots.

NewBlot IR works well for removal of all IRDye secondary antibodies, and may be suitable for removal of other dye-conjugated secondary antibodies.

NewBlot IR does not require hazardous shipping, unlike many other stripping buffers.

Note: NewBlot IR Stripping Buffer is not recommended for loading amounts over 30 µg.

Choosing the Right Stripping Buffer

Specifications

NewBlot IR Stripping Buffer is supplied as a 5X concentrated solution. Each 100 mL bottle is sufficient for up to 3000 cm2 or approximately fifty 7 × 8.5 cm Odyssey® nitrocellulose membranes.

Example Data

Strip and reprobe nitrocellulose or PVDF membranes
Stripping and reprobing nitrocellulose or PVDF membranes effectively with NewBlot IR Stripping Buffer. EGFR and phospho-ERK levels were compared in EGF-stimulated (+) and non-stimulated (-) A431 lysate (1 µg total protein). The membrane was probed with mouse anti-EGFR, mouse anti-pERK1/2, and Β-tubulin rabbit polyclonal (P/N 926-42211), then detected with IRDye 800CW Goat anti-Mouse (P/N 926-32210) and IRDye 680RD Goat anti-Rabbit (P/N 926-68071). The blot was scanned with an Odyssey CLx Imaging System (Original blot). The blot was then stripped with NewBlot IR Stripping Buffer (P/N 928-40028), and scanned again (Strip #1). The blot was detected with the same primary and secondary antibodies and scanned again (Reprobe #1). The process was repeated 2 more times (Strip and Reprobe #2-3). Image display settings for all stripped and reprobed images are identical to the original image.

Factors Affecting Efficiency when using NewBlot IR Stripping Buffer

Recommended Standard Conditions: 1X NewBlot IR for 20 minutes at room temperature.

Below are key factors that affect stripping efficiency with NewBlot IR on Odyssey nitrocellulose or Immobilon®-FL PVDF membranes.

For optimal stripping results, follow the optimization guidelines in NewBlot IR Stripping Buffer pack insert.

Amount of time blot is in stripping buffer

Increasing the stripping time may increase stripping efficiency. However, it may also remove target proteins 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.

Note: NewBlot IR Stripping Buffer is not recommended for loading amounts over 30 µg.

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 or temperature will reduce stripping efficiency and is not recommended for NewBlot IR Stripping Buffer. Use the material at a 1X concentration at room temperature for optimal results.

Selected P/N: 928-40028

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