Abcam IP 免疫沉澱實驗產品特輯

Abcam IP 免疫沉澱實驗產品特輯

免疫沉澱 (immunoprecipitation, IP) 是一種以抗體作為核心基礎的實驗技術,可以將目標蛋白質從細胞或組織樣本中分離出來 (isolate),也可以用來富集 (enrich) 特定的微量蛋白質。在進行 IP 實驗時,流程可以大致區分為三個部分:(1) 將專一性抗體與樣本混合,捕捉目標蛋白質;(2) 加入 Protein A 或 G 親和性層析膠體(或磁珠),形成“抗原-抗體-膠體”複合物;(3) 自膠體上沖提出目標蛋白質,並以西方墨點法 (western blot, WB) 或質譜儀 (mass spectrometry, MS) 進行分析。

免疫沉澱 (immunoprecipitation, IP) 實驗原理示意圖

免疫沉澱 (immunoprecipitation, IP) 實驗原理示意圖

以 IP 實驗為基礎,還延伸出可以用來研究蛋白質間交互作用的免疫共沉澱法 (co-immunoprecipitation, co-IP)、研究蛋白質與 Genomic DNA 交互作用的染色質免疫沉澱法 (chromatin immunoprecipitation, ChIP) 以及研究蛋白質與 RNA 交互作用的 RNA 免疫沉澱法 (RNA immunoprecipitation, RIP)。

為協助您更快更好地完成 IP 實驗,Abcam 團隊精心打造了多項 IP 實驗工具,從 IP 實驗用一級抗體與二級抗體、到純化膠體與 IP Kit 等超過 8,100 種產品選擇。以下就為您整理出深受全球科研用戶喜愛與信賴的【IP 實驗明星商品】,同時也歡迎您透過 IP ProtocolAdvanced Immunoprecipitation 頁面進一步了解實驗注意事項、試劑挑選準則等實用資訊。如果懶得爬文,也可以將手邊各種 IP 實驗疑難雜症直接交給伯森業務專員解決哦👌!

IP 實驗用一級抗體

  • 效能通過實際 IP 實驗驗證:一個好的 IP 實驗用一級抗體,不僅要能夠專一性地辨識天然原始構形 (native conformation) 狀態下的目標蛋白質,還必須具有足夠高的親和性才能滿足 IP 實驗的要求。Abcam IP 實驗用一級抗體通過實際 IP 實驗驗證,確保能夠提供精確優質的實驗結果。

Immunoprecipitation (IP) validation - Recombinant Anti-PD-L1 antibody [73-10]

Immunoprecipitation (IP) validation - Recombinant Anti-PD-L1 antibody [73-10] (ab228415). PD-L1 was immunoprecipitated from 0.35 mg of NCI-H1975 (human non-small cell lung cancer cell line) whole cell lysate with Anti-PD-L1 antibody [73-10] (ab228415) at 1/30 dilution. Western blot was performed from the immunoprecipitate using Anti-PD-L1 antibody [73-10] (ab228415) at 1/1,000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution. Lane 1: NCI-H1975 whole cell lysate 10 µg (Input). Lane 2: ab228415 IP in NCI-H1975 whole cell lysate (+). Lane 3: Rabbit monoclonal IgG - Isotype Control (ab172730) instead of Anti-PD-L1 antibody [73-10] (ab228415) in NCI-H1975 whole cell lysate (-). Blocking/Dilution buffer: 5% NFDM/TBST.

  • Tag 抗體滿足 co-IP 實驗需求:在進行 co-IP 實驗時,經常會先使用重組蛋白質技術大量表現出帶有特殊標籤序列(如 His tag, GST tag, Myc tag...)的目標蛋白質,再以該融合蛋白質與 Tag 抗體進行 co-IP 實驗。以下為您整理出適用於 IP 實驗的 Tag 抗體明星商品
明星商品:Tag Antibodies for IP
Tag Type Product Name Cat. No.
His tag® Anti-6X His tag® antibody [HIS.H8] ab18184
Recombinant Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204
GST tag Anti-GST antibody [3G10/1B3] ab92
Recombinant Anti-GST antibody [EPR4236] ab111947
Myc tag Anti-Myc tag antibody [9E10] ab32
Anti-Myc tag antibody [Myc.A7] ab18185
HA tag Anti-HA tag antibody - ChIP Grade ab9110
Recombinant Anti-HA tag antibody [EPR22819-101] ab236632
FLAG® tag Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [FG4R] ab125243
Recombinant Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] ab205606
  • 同型對照抗體 (isotype controls) 協助您確認實驗結果的專一性:在進行 IP 實驗時,除了 No Antibody (不加 IP 一抗) 對照組以外,建議同時設立 Isotope Control 對照組,以協助確認 WB 或 MS 檢測結果的專一性。選擇 Isotype Control 抗體時,需選用與 IP 一抗具有相同宿主物種、抗體亞型與標定分子(例如 Biotin, FITC 等)的抗體。

IP 實驗用二級抗體(解決 WB 檢測結果中的輕鏈與重鏈干擾問題)

在將目標蛋白質從膠體上沖提下來時,IP 一抗往往也會跟隨著被沖提下來。若用來檢測目標蛋白質的 WB 一抗剛好和 IP 一抗有著同樣的宿主物種來源,WB 二抗在辨識 WB 一抗時就會連同 IP 一抗一起辨識,最終導致 WB 檢測結果多出兩條由變性的 (denatured) IP 一抗輕鏈 (light chain, 約 25 kDa) 與重鏈 (heavy chain, 約 50 kDa) 形成的條帶 (bands)。這兩條 bands 不僅會造成 WB 結果判讀不易,有時甚至會完全遮蓋掉目標蛋白質的訊號!為了解決這個問題,Abcam 團隊研發出 IP 實驗用二級抗體,僅需在 WB 檢測時,以 IP 實驗用二抗取代原本的 WB 二抗,就能徹底擺脫輕鏈與重鏈干擾,得到更清晰精確的 WB 檢測結果。目前提供以下兩種系列的 IP 實驗用二抗產品:

  • VeriBlot 二級抗體:僅會辨識天然、非變性狀態的抗體,因此樣本中已經經過加熱及 SDS 處理的變性 IP 一抗,就不會被 VeriBlot 二級抗體所辨識,在 WB 檢測結果中也就不會看到輕鏈與重鏈的 bands。所有分子量大小的目標蛋白質皆可適用。
  • Light chain specific 輕鏈專一性二級抗體:僅會辨識天然、非變性狀態的抗體輕鏈。目標蛋白質分子量 >30 kDa 時適用。
明星商品:Secondary Antibodies for IP
Type Product Name Conjugate Cat. No.
VeriBlot VeriBlot for IP Detection Reagent
Reactivity: Rabbit total IgG, Bovine IgG₂, Goat IgG₂, Human IgG₁, IgG₂, IgG₄, Mouse IgG₂a, IgG₂b, IgG₃, Rat IgG₂c, Sheep IgG₂
HRP ab131366
Anti-mouse IgG for IP HRP ab131368
Anti-mouse IgG for IP Biotin ab131367
Mouse Anti-goat IgG for IP HRP ab157532
Light chain specific Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain HRP ab99697
Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain AP ab99696
Mouse monoclonal [KT102] Anti-Rat kappa light chain antibody HRP ab170326
Mouse monoclonal [JDC-12] Anti-Human lambda light chain HRP ab99811

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366). Immunoprecipitating IRAK2 in human HEK293 whole cell lysate. 1000µg of cell lysate was incubated with Anti-IRAK2 antibody (ab6148) (1 µg/mg) and matrix (Protein G) for 16 hours at 4°C. For western blotting a HRP-conjugated Veriblot for IP Detection Reagent (ab131366) (1/10000) was used to confirm successful immunoprecipation.

Immunoprecipitation - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)

Immunoprecipitation - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697). LAP2 alpha was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to LAP2 alpha (ab5162) and 50µl of protein G magnetic beads (lane 1). The antibody was incubated with the Protein G beads for 10min under agitation. No antibody was added to the control (lane 2). Hela whole cell extract diluted in RIPA buffer was added to each sample and incubated for 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab5162. Secodary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Bands: LAP2 alpha, 83kDa.

抗體純化膠體

Protein A 與 Protein G 層析膠體是最常使用的抗體純化膠體,其次為 Protein L 層析膠體。不同於 Protein A 與 Protein G 是和抗體的 Fc 區域結合,Protein L 則是和抗體的 Kappa (κ) 輕鏈結合,因此相較於 Protein A 或 G 膠體,Protein L 膠體可以辨識純化的抗體種類範圍更廣,包含 IgG 抗體、IgM 抗體、IgA 抗體、IgE 抗體、IgD 抗體、Fab 抗體與 ​scFv​ 抗體等。Abcam 提供多款層析膠體與預注管柱產品選擇,您可參考【Protein A, Protein G, Protein L 抗體結合力對照表】依據您 IP 一抗的抗體種類與亞型選擇適合的產品,或洽詢伯森業務專員

明星商品:Antibody Purification Resins and Prepacked Columns
Type Product Name Binding Capacity* Cat. No.
Protein A Protein A Resin - Amintra >40 mg IgG/ml ab270308
Protein A Agarose (High Affinity) >35 mg IgG/ml ab193255
Protein A Magnetic Beads >25 mg/ml ab214286
Protein A Agarose >20 mg IgG/ml ab193254
Protein A Sepharose® >16 mg IgG/ml ab193256
Protein A Sepharose® Column >16 mg IgG/ml ab193257
Protein G Protein G Resin - Amintra >30 mg IgG/ml ab270309
Protein G Agarose (High Affinity) >30 mg IgG/ml ab193258
Protein G Sepharose® >20 mg IgG/ml ab193259
Protein G Sepharose® Column >20 mg IgG/ml ab193260
Protein L Protein L Sepharose® >10 mg IgG/ml ab193261
Protein A/G Protein A/G Sepharose® >10 mg IgG/ml ab193262
Protein A/G Sepharose® Column >10 mg IgG/ml ab193263
Protein A/G/L Protein A/G/L Sepharose® >10 mg IgG/ml ab193264
Protein A/G/L Sepharose® Column >10 mg IgG/ml ab193265

*Human or rabbit IgG

IP Kit

便利的免疫沉澱實驗試劑套組,從樣本製備過程所需的裂解緩衝液(lysis buffer)、蛋白酶抑制劑混合物 (protease inhibitor cocktails),到純化膠體與沖洗液 (wash buffer),一應俱全!

明星商品:IP Kit
Product Name Pack Size Cat. No.
Immunoprecipitation kit 25 tests ab206996

Immunoprecipitation - Immunoprecipitation kit (ab206996)

Immunoprecipitation - Immunoprecipitation kit (ab206996). Co-immunoprecipitation experiments indicate IKKε does not interact with NIK or IKKε. Immunoprecipitation was performed on MDA MB 468 cells using the Abcam immunoprecipitation kit (ab206996), according to manufacturer's instructions. Briefly, non-denaturing lysis buffer was used to collect 300 µg of cell lysate was incubated overnight with 3 µg/ml of either control rabbit IgG or IKKε rabbit polyclonal antibody (ab7891). Antibody bound proteins were captured using protein A/G sepharose beads, eluted, and analyzed via western blot. IMAGE © BMC Cancer. 2018 May 25;18(1):595. PMID: 29801480. Fig. 4e.

樣本製備試劑

Abcam 提供多款裂解緩衝液、蛋白酶抑制劑混合物與蛋白質定量產品,讓您從容地完成 IP 實驗的樣本製備工作。

明星商品:Sample Preparation Reagents for IP
Type Product Name Cat. No.
Lysis Buffer Cell Lysis Buffer ab152163
Native lysis Buffer ab156035
10X RIPA Buffer ab156034
Protease Inhibitor Cocktails Protease Inhibitor Cocktail ab271306
Protease Inhibitor Cocktail EDTA Free ab270055
Protease and Phosphatase Inhibitor Cocktail ab201119
Protease and Phosphatase Inhibitor Cocktail (EDTA-free) ab201120
Protein Quantification Bradford Reagent ab119216
BSA protein standards ab270701

完整產品資訊與最新活動訊息,歡迎洽詢 Abcam 台灣代理 — 伯森生技。任何 IP 或 WB 實驗問題,都歡迎隨時與我們一同討論喔~ (👨‍💻伯森生技線上線下聯絡資訊

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