上層膠溶液及下層膠溶液都已預配好,僅需新鮮加入APS及TEMED即可快速鑄膠。
蛋白質電泳以及西方墨點法
蛋白質電泳以及西方墨點法是常用的技術,超過8成以上的蛋白質研究實驗室都會使用,也是文獻發表通常必備的數據。聚丙烯醯胺凝膠電泳(簡稱PAGE)是膠體電泳的一種,此項技術的原理,是利用檢體中蛋白質分子量大小的不同,使蛋白質分子在電泳凝膠中分離。
當蛋白質電泳與膜轉漬技術、抗原抗體反應、以及成像技術結合起來,就是分子生物學實驗室常用的西方墨點法(western-blotting)。
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- Sample buffer
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- Transfer buffer
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聚丙烯醯胺(Polyacrylamide)凝膠化學
聚丙烯醯胺凝膠電泳(Polyacrylamide gel electrophoresis)簡稱 PAGE,PAGE 使用不連續緩衝系統,在凝膠中的緩衝離子與跑膠緩衝液中的離子不同,這兩種離子之間的電泳移動率差異形成了蛋白質穿過的移動電壓梯度。
「Tris-Glycine 凝膠化學」是最常用的 PAGE 系統,它使用由 Tris-HCL 組成的凝膠以及由 Tris base 和 Glycine 組成的跑膠緩衝液。Tris-Glycine 系統在強鹼性環境中運作,可能導致我們不希望發生的蛋白質修飾(例如:脫氨作用和烷基化作用),因此導致蛋白質條帶可能會扭曲或降低解析度,凝膠的保存期限也比較有限(隨著時間發生水解)。
相較之下,「Bis-Tris 凝膠化學」系統在凝膠緩衝液中使用 Bis-Tris 和 HCL,在跑膠緩衝液中使用 MOPS 或 MES,讓整個系統在中性 pH 值下運作,可以大幅避免蛋白質發生修飾、提高蛋白質穩定性,因此可以提供更清晰的蛋白質條帶解析度和準確性。同時,也具有更快的跑膠速度、更長的保存期限,也可以靈活地與MOPS或MES跑膠緩衝液配合使用。因此,Bis-Tirs 系統的好處是:具有更清晰的蛋白質條帶解析度以及準確性,更快的跑膠速度,更長的保存期限,可以靈活搭配 MES 或 MOPS 兩種 Running Buffer 做使用。
TurboMix™ Bis-Tris PAGE Casting Kit快速鑄膠套組
「上層膠溶液」與「下層膠溶液」均已預配好,僅須加入新鮮的 APS 及 TEMED!下層膠溶液可自行稀釋至需求之濃度。
Bis-Tris系統具有優秀數據解析度,與一般 Tris-Glycine 系統相比,Bis-Tris 維持系統 pH 中性,蛋白條帶更清晰、數據解析度更高。
TurboMix™ Bis-Tris PAGE Casting Kit 操作流程
- 製備 10% APS 溶液
- 加水將 Resolving Mix 稀釋至需求的濃度後,加入 APS 及 TEMED
- 將 APS 及 TEMED 加入 Resolving Mix
- 將 Resolving Mix 加入鑄膠台玻片中
- 將 Stacking Mix 加入鑄膠台玻片中並插入齒梳,等待凝結就完成囉!
觀看TurboMix™操作影片
Bis-Tris系統搭配之buffer說明
【LDS sample buffer】Bis-Tris系統的pH值為中性,可以提高跑膠過程的蛋白質穩定性。使用LDS Sample Buffer,從收樣本的那一刻起,即以中性pH值溫和保護蛋白質。
【Bis-Tris running buffer】兩款running buffer適合分離不同分子量的目標蛋白質:MES適合分離小分子量蛋白,MOPS適合分離大分子量蛋白。
同一片凝膠,搭配不同的running buffer使用,可著重分離偏大或偏小分子量的目標蛋白族群。
【Bis-Tris transfer buffer】
Bis-Tris系統的pH值為中性,可以提高跑膠過程的蛋白質穩定性。使用Bis-Tris transfer buffer,讓轉漬過程也持續溫和保護蛋白質。
Materials
Product Number |
Product Description |
TMKIT |
TurboMix™ Bis-Tris Polyacrylamide Gel Casting Kit |
TMRES-216ML |
TurboMix™ Resolving Solution, 216 mL |
TMSTK-120ML |
TurboMix™ Stacking Solution, 120 mL |
MPTA |
Adapters for mPAGE® gels when using XCell SureLock® and other compatible electrophoresis tanks |
MPMES |
MES SDS running buffer powder for mPAGE® Bis-Tris gelspack of 5 ea (Each packet makes 1 liter of running buffer) |
MPM0PS |
MOPS SDS running buffer powder for mPAGE® Bis-Tris gelspack of 5 ea (Each packet makes 1 liter of running buffer) |
MP41G10 |
mPAGE® 4-12% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 10 well |
MP41G12 |
mPAGE® 4-12% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 12 well |
MP41G15 |
mPAGE® 4-12% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 15 well |
MP42G10 |
mPAGE® 4-20% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 10 well |
MP42G12 |
mPAGE® 4-20% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 12 well |
MP8W10 |
mPAGE® 8% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 10 well |
MP8W12 |
mPAGE® 8% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 12 well |
MP8W15 |
mPAGE® 8% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 15 well |
MP81G10 |
mPAGE® 8-16% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 10 well |
MP81G12 |
mPAGE® 8-16% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 12 well |
MP81G15 |
mPAGE® 8-16% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 15 well |
MP10W10 |
mPAGE® 10% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 10 well |
MP10W15 |
mPAGE® 10% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 15 well |
MP10W12 |
mPAGE® 10% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 12 well |
MP12W10 |
mPAGE® 12% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 10 well |
MP12W12 |
mPAGE® 12% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 12 well |
MP12W15 |
mPAGE® 12% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 12 well |
MP41G12TR1 |
mPAGE® Trial Kitincludes 2 4-12% Bis-Tris precast gels, 10x8 cm, 12-well, MOPS SDS powder, makes 1L of running buffer, 2 tank adapters |
MP42G12TR1 |
mPAGE® Trial Kitincludes two 4-20% Bis-Tris precast gels, 10x8 cm, 12-well, MOPS SDS powder, makes 1L of running buffer, 2 tank adapters |
MP10W12TR1 |
mPAGE® Trial Kitincludes two 10% Bis-Tris precast gels, 10x8 cm, 12-well, MOPS SDS powder, makes 1L of running buffer, 2 tank adapters |
MP41G12TR2 |
mPAGE® Trial Kitincludes two 4-12% Bis-Tris precast gels, 10x8 cm, 12-well, MES SDS powder, makes 1L of running buffer, 2 tank adapters |
MP10W12TR2 |
mPAGE® Trial Kitincludes two 10% Bis-Tris precast gels, 10x8 cm, 12-well, MES SDS powder, makes 1L of running buffer, 2 tank adapters |
MP12W12TR2 |
mPAGE® Trial Kitincludes two 12% Bis-Tris precast gels, 10x8 cm, 12-well, MES SDS powder, makes 1L of running buffer, 2 tank adapters |
MPC0 |
Tool for operning mPAGE® gel cassettes |
MPTRB |
Transfer buffer powder for use with mPAGE® Bis-Tris gelspack of 10 ea (Each packets makes 1 liter of transfer buffer at 1X) |
MPBD |
When running a single gel, the buffer dam can be used to occupy space in the electrophoresis tank normally taken by another gel. |
A3656 |
5-Aza-2′-deoxycytidine≥97% |
T22500 |
N,N,N′,N′-TetramethylethylenediamineReagentPlus®, 99% |
43816 |
DL-Dithiothreitol solutionBioUltra, for molecular biology, ~1 M in H2O |
63689 |
2-MercaptoethanolBioUltra, for molecular biology, ≥99.0% (GC) |
MPSTD4 |
mPAGE® Color Protein Standard |
MPSTD3 |
mPAGE® Unstained Protein Standard |
G1041 |
EZBlue™ Gel Staining Reagent |
SCT145 |
EZFluor™ 1-step Fluorescent Protein Gel Stain (1L)Fluorescent protein gel stain that requires only a single 5-30 minute staining step without fixation. |
SCT147 |
EZFluor™ UV 1-step Fluorescent Protein Gel Stain (1L)Fluorescent protein gel stain that requires only a single 5-30 minute staining step without fixation. |
PROTSIL1 |
ProteoSilver™ Silver Stain Kit |
Reference
1.Robinson DR, Watts NR, Coombs DH. 1988. Heat cleavage of bacteriophage T4 gene 23 product produces two peptides previously identified as head proteins. J Virol. 62(5):1723-1729. http://dx.doi.org/10.1128/jvi.62.5.1723-1729.1988