NewBlot PVDF 剥离缓冲液专为 IRDye® 二抗配制,NewBlot PVDF 仅适用于 PVDF 膜

Reagents > NewBlot™ PVDF Stripping Buffer for PVDF Membranes,用于 PVDF 膜的 NewBlot™ PVDF 剥离缓冲液

NewBlot™ PVDF Stripping Buffer for PVDF Membranes

NewBlot PVDF stripping buffer is specially formulated for IRDye® secondary antibodies including IRDye 800CW, IRDye 680RD, and IRDye 680LT.

NewBlot PVDF is intended for PVDF membranes only and is ideal for removal of secondary antibody.

Choosing the Right Stripping Buffer

NewBlot PVDF 剥离缓冲液专为 IRDye® 二抗配制，包括 IRDye 800CW、IRDye 680RD 和 IRDye 680LT。

NewBlot PVDF 仅适用于 PVDF 膜，是去除二抗的理想选择。

选择正确的剥离缓冲液

Specifications

NewBlot PVDF Stripping Buffer is supplied as 5X concentrated solutions. Each 100 mL bottle is sufficient for up to 3000 cm2 or approximately fifty 7 x 8.5 cm Millipore® Immobilon®-FL PVDF membranes.

NewBlot PVDF stripping buffer contains hazardous materials and additional shipping costs will be applied.

NewBlot PVDF 剥离缓冲液以 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 Millipore® Immobilon®-FL PVDF 膜。

NewBlot PVDF 剥离缓冲液含有有害物质，将产生额外的运输费用。

Example Data

NewBlot™ PVDF Stripping Buffer for PVDF Membranes
Figure 1. Example of stripping and reprobing with NewBlot PVDF Stripping Buffer. (A) Invitrogen EPAGE-96 6% gel loaded with replicate A431 lysate samples and detected with anti-Syk (IRDye 680, red bands) and anti-β-Tubulin (IRDye 800CW, green bands). (B) Stripped with NewBlot-PVDF Stripping Buffer for 5 min at room temperature. (C) Blot was reprobed with anti-Syk and anti-β-tubulin antibodies. All blots have been scanned at 169 µm resolution and scan intensity of 6 on the Odyssey Classic Imager.
图 1. 使用 NewBlot PVDF 剥离缓冲液进行剥离和重新检测的示例。 (A) Invitrogen EPAGE-96 6% 凝胶加载重复的 A431 裂解物样品，并用抗 Syk（IRDye 680，红色条带）和抗 β-微管蛋白（IRDye 800CW，绿色条带）检测。 (B) 在室温下用 NewBlot-PVDF 剥离缓冲液剥离 5 分钟。 (C) 用抗 Syk 和抗β-微管蛋白抗体重新探测印迹。 所有印迹都在 Odyssey Classic Imager 上以 169 µm 的分辨率和 6 的扫描强度进行扫描。
NewBlot™ PVDF Stripping Buffer for PVDF Membranes
Figure 2. Images showing an example of Western blot stripping optimization with NewBlot PVDF Stripping Buffer. (A) Initial Western blot, showing EGFR detected with IRDye 800CW goat anti-mouse and β−Actin detected with IRDye 680LT goat anti-rabbit. (B) After stripping with standard stripping Procedure. (C) After stripping for an additional 20 min. (D) After additional 5 min stripping with the addition of SDS. (E) After 20 minutes total stripping time in NewBlot PVDF + SDS. (F) Reprobe image showing ERK2 detected with IRDye 680LT goat anti-mouse and β−Tubulin detected with IRDye 800CW goat anti-rabbit.

Factors Affecting Efficiency when Using NewBlot PVDF Stripping Buffer

Recommended standard conditions: 1X NewBlot PVDF for 20 min at room temperature.

Below are factors that affect stripping efficiency with NewBlot PVDF on PVDF membranes. Figure 2 above shows the effects of incubation time and addition of SDS on stripping efficiency. These blots and protein targets were not sufficiently stripped using the standard Procedure, and optimization was performed.

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

推荐的标准条件：1X NewBlot PVDF 在室温下 20 分钟。

以下是影响使用 NewBlot PVDF 在 PVDF 膜上剥离效率的因素。 上面的图 2 显示了孵育时间和添加 SDS 对剥离效率的影响。 使用标准程序没有充分剥离这些印迹和蛋白质靶标，并进行了优化。

为获得最佳剥离结果，请遵循 NewBlot PVDF 剥离缓冲液包插入中的优化指南。

Amount of time blot is in stripping buffer

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.

即使在最严格的剥离条件下，由于样品上样量、抗体亲和力/亲合力和靶蛋白丰度，荧光信号也可能无法完全去除。

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.

Detergent may help remove fluorescent signal

If fluorescent signal remains, try longer incubation times first, followed by addition of SDS.

Temperature used for stripping

If fluorescent signal remains after the above steps, incubation in stripping buffer may be carried out at 37 °C using a water bath or incubator.  This should only be attempted if the above optimization steps are unsuccessful.

如果在上述步骤后仍有荧光信号，则可以使用水浴或培养箱在 37 °C 下在剥离缓冲液中进行孵育。 仅当上述优化步骤不成功时才应尝试此操作。

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