OneStep Blocker封閉&抗體增強溶液 | BIO-HELIX貨號 BS001-B500ML

OneStep Blocker 緩衝液一個步驟同時完成封閉(blocking)、一抗和二抗雜交(hybridization)等三個實驗步驟，OneStep Blocker 同時也能增強 HRP（horseradish peroxidase）或 AP（alkaline phosphatase, 鹼性磷酸酶）底物產生的signal。在西方分析(Western analysis)中既是阻斷劑(blocker)又是增強劑(enhancer)。不含蛋白質配方(Protein free)大幅減少 ECL 檢測中常見的非特異性信號，降低背景值。適用於所有PVDF & NC membrane。

Mix OneStep Blocker, 1°Ab and 2° Ab with membrane together, that’s all.

OneStep Blocker is a blocking solution for Western blot analysis. This OneStep Blocker buffer not only provides blocking, primary and secondary antibody hybridization in one step but also enhances the signal developed with HRP (horseradish peroxidase) or AP (alkaline phosphatase) substrates. It, therefore, serves as both blocker and enhancer in Western analysis. With the 3-in-1 step procedure, OneStep Blocker is a time and labor economic solution for the time consuming and laborious Western procedure.

Features of OneStep Blocker – Western blocking solution and a signal enhancer (Protein free)

• 3 steps in one: Block the membrane and dilute 1o  Ab & 2o  Ab in one step.
• Enhance antibody signal: It shows a two- to five-fold increase in signal intensity for most protein targets, enabling much less protein to be detected with the same substrate and method.
• Enhance time-saving: It saves at least 2 hours in the antibody detection process during the Western Blot, with only one hour needed.
• Universal antibody diluent: Ready-to-use dilution buffer for most of 1o  Ab & 2o  Ab.
• No blocking step needed: Just immerse the membrane in the OneStep Blocker solution with your antibodies. That’s all.
• Effective with any ECL: After the antibody detection process, the signal can be developed with both HRP (horseradish peroxidase) and AP (alkaline phosphatase) substrates.
• Less hands-on steps: No 3 wash steps are required, meaning no need to transfer the membrane in&out of the container.
• Compatible with PVDF & NC membrane: Regardless of the pore size, the OneStep Blocker minimizes the background from non-specific protein binding by antibodies.
• Improve protein detection: Improve the binding process of target proteins, so that specific antibodies can bind more effectively.
• Protein free: Reduces overall background and minimizes non-specific signals often seen with ECL detection.

 

1. Western blot 轉移後，將 PVDF 或 NC 膜浸入 PBST 緩衝液中 5 分鐘。
2. 用適量的 OneStep Blocker 稀釋一抗和二抗。

——2.1 例如，當一抗和二抗的稀釋倍數為 1：10,000 時，將 2μl 一抗加入 10ml OneStep Blocker（第 1 管）中，然後將 2μl 二抗加入另外 10ml OneStep阻滯劑（第

2  管）。

——2.2 通過來回inverting將每個管內的抗體-OneStep Blocker 溶液徹底混合。

——2.3 先將一抗-OneStep Blocker 溶液倒入準備好的容器中，然後將二抗-OneStep Blocker 溶液加入同一個容器中。

3. 立即將膜在抗體-OneStep Blocker 溶液中室溫孵育 1-2 小時，同時輕輕攪拌。請注意，一抗和二抗混合後，膜需要在 10 分鐘內立即浸入混合物中，以獲得最佳性能。
4. 用 PBST/TBST 振盪洗滌膜 3 次。
5. 排出過多的洗滌緩衝液並立即使用 ECL 或比色系統進行顯像。

注意事項

一抗和二抗混合後，需要在 10 分鐘內立即將膜浸入混合物中，以獲得最佳性能。二抗的稀釋度應至少為 1:10,000 或更高。使用高濃度二抗時會觀察到更高水平的背景噪音。不要在 OneStep Blocker 中孵育膜超過 4 小時，以避免高背景。尤其不推薦過夜孵育(O/N)。OneStep Blocker 溶液中混合的一抗/二抗可在 3 天內重複使用。隨著存儲時間或重複性的增加，增強效果可能會逐漸減弱。將混合溶液冷藏。對於關鍵實驗或強信號，需要新鮮製備抗體 OneStep Blocker 溶液。抗體濃度過高或孵育時間過長，會引起高背景。當出現過多背景時，請嘗試以下方法：

(a) 降低/優化一抗和/或二抗濃度。

(b) 使用斑點印跡測試優化抗體濃度。

(c) 減少/優化孵育時間。