The Optimization Concept of Western Blotting

Transcription

1 2013/8/12 The Optimization Concept of Western Blotting 行易實業 ( 股 ) 公司產品專員楊惠而 (Emma) 2013/8/12 1 Western blotting workflow Sample preparation Gel electrophoresis Transfer Antibody probing Detection Imaging Analysis 2013/8/12 2 1

2 2013/8/12 Problems You May Meet in WB Problem: Proteins with similar masses migrate together 1. Gel concentration Variable concentration Gradient gel Gel 10% 12% 4-12% 8-16% 4-20% Concentration gradient gradient gradient -electrophoresis /8/12 3 Problems You May Meet in WB Problem: Proteins with similar masses migrate together 1. Gel concentration 2. SDS-PAGE buffer system Tris-glycine buffer: Mr >20KDa Tris-Tricine buffer: Mr <20KDa 10% T, 3% C gels a: Tris-Tricine b: Tris-Glycine Nature Protocols 1, (2006) 2013/8/12 4 2

3 2013/8/12 Problems You May Meet in WB Problem: Poor transfer 1. Confirmation of protein transfer to the membrane Gel staining: Coomassie blue Membrane staining: PonceauS 2013/8/12 5 Problems You May Meet in WB Problem: Poor transfer 1. Confirmation of protein transfer to the membrane 2. Protein blow-through Membrane pore size 0.2 um membrane: Mr <20KDa 0.45um membrane: Mr >20KDa Protein-Membrane affinity 2013/8/ um SDS-GEL 0.2um 0.2um 6 3

4 2013/8/12 Problems You May Meet in WB Problem: Poor transfer 1. Confirmation of protein transfer to the membrane 2. Protein blow-through Membrane pore size Protein-Membrane affinity Brand M Brand M Hybond-P Hybond-P 2013/8/12 7 Problems You May Meet in WB Problem: Poor transfer 1. Confirmation of protein transfer to the membrane 2. Protein blow-through Membrane pore size Protein-Membrane affinity M 牌 : R 2 = GE: R 2 = /8/12 8 4

5 2013/8/12 Problems You May Meet in WB Problem: Low specificity with high background 1. Blocking inefficient 2013/8/12 9 Problems You May Meet in WB Problem: Low specificity with high background 1. Blocking inefficient Filter blocking buffer (0.22um filter) Change blocking buffer recommended Protein concentration (W/V) BSA 0.2-5% Non-fat milk 3-5% non-compatible application biotin/streptavidin system glycoprotein detection phosphoprotein detection AmershamECL Prime Blocking 2-5% Agent Casein 1% phosphoprotein detection Fish Gelatin 2-10% 2013/8/12 Serum 1-5% 10 5

6 2013/8/12 Problems You May Meet in WB Problem: Low specificity with high background 1. Blocking inefficient 2. Primary antibodies selection Validated for Western blotting applications Sensitivity: pab >mab; Specificity: mab >pab Antibodies can be affinity-purified using immobilized antigenor immobilized Protein G or Protein A 3. Timing and wash step 2013/8/12 11 Problems You May Meet in WB Problem: Low specificity with high background Strategy for antibody purification Antigen yes no Antigen-coupling method Protein A/G matrix Mr <5000Da Mr >5000Da Volume >2mL Volume <2mL NHS-activated matrix CNBr-activated matrix Sepharose beads Magnetic beads 2013/8/

7 2013/8/12 Problems You May Meet in WB Problem: 1. White band on film(burn out) Reduce 1Ab/2Ab concentration Use less sensitive ECL substrate 2. No signal/weak signal Increase 1Ab/2Ab concentration Change ECL substrate: More sensitive Enhance intensity: Increase exposed time CCD is better than Film 3. Un-repeated experiment Change ECL substrate: Long duration time 2013/8/12 13 Problems You May Meet in WB Problem: 1. White band on film(burn out) Reduce 1Ab/2Ab concentration Use less sensitive ECL substrate 2. No signal/weak signal CCD Increase 1Ab/2Ab concentration Change ECL substrate: More sensitive Enhance intensity: Increase exposed time CCD is better than Film 3. Un-repeated experiment Change ECL substrate: Long duration time Film /8/

8 2013/8/12 Problems You May Meet in WB Problem: 1. White band on film(burn out) Reduce 1Ab/2Ab concentration Use less sensitive ECL substrate 2. No signal/weak signal Increase 1Ab/2Ab concentration Change ECL substrate: More sensitive Enhance intensity: Increase exposed time CCD is better than Film 3. Un-repeated experiment Change ECL substrate: Long duration time 2013/8/12 15 Problems You May Meet in WB Problem: 1. White band on film(burn out) 2. No signal/weak signal 3. Un-repeated experiment Solution: 1. Optimizing 1Ab/2Ab concentration 2. ECL substrate with suitable sensitivity 3. ECL substrate with longer duration time 4. Enhance intensity: increase exposure time 2013/8/

9 2013/8/12 產品偵測類型 Amersham ECL 冷光偵測 Amersham ECL Prime 冷光偵測 Amersham ECL Select 冷光偵測 適用偵測範圍 高 / 中表現量蛋白質 中 / 低表現量蛋白質 中 / 非常低表現量蛋白質 適用膜種類 Hybond ECL(NC) Hybond-P(PVDF) Hybond ECL(NC) Hybond-P(PVDF) Hybond ECL(NC) 特色 最早用於 western blotting 的偵測試劑之一 超過 3000 篇文獻使用此偵測試劑 節省抗體 : 高度稀釋一抗 / 二抗 超高持久度 : 適合大量實驗偵測 節省抗體 : 高度稀釋一抗 / 二抗 超高持久度 : 適合大量實驗偵測 偵測方式 Hyperfilm (X-ray film) ImageQuant LAS series X-ray film is also an option ImageQuant LAS series X-ray film is also an option 2013/8/12 17 AmershamECL Select fit most WB experiments Step1 Step2 Step3 Step4 Step5 Equilibrate reagents for 20mins Mix A and B in a ratio of 1:1 Apply mixture of ECL into a series dilution of 2 nd Ab Incubate for 5 mins Detect signals 1x Select 2x Select 4x Select 30 th min 2Ab 8x Select 1x BrandX 2x BrandX 2013/8/

10 2013/8/12 WB Total Healthcare Sample preparation Gel electrophoresis Transfer Antibody probing Detection Imaging Analysis 2013/8/12 19 Useful Resources WB Handbooks WB microsite ( Request (For FREE) Handbook_Western blotting principle and methods 2013/8/

11 2013/8/12 Appreciate your attention! Any questions? 有任何技術問題, 歡迎與行易連絡! 客服專線 (02) 網頁 /8/

12 ImageQuant LAS 4000/4000mini Biomolecular imagers 李宗樹 奇異亞洲醫療設備股份有限公司 GE Healthcare Life Sciences Source: Fujifilm 1 /

13 NEW biomolecular imagers ImageQuant LAS 4000 ImageQuant LAS 4000 mini Typhoon FLA 9000 Typhoon FLA 7000 LAS4000 in Olympics Games 4 / 2 /

14 Film vs. LAS system Film LAS system Space dark room needed on bench Consumables film needed no film Exposure type manual precision auto/maunal precision, increment, repetition, program Quantitation procedure Quantitation dynamic range exposure -> develop-> scan -> analyze exposure->analyze 1-2 orders 3-4 orders Sensitivity pg level pg level Environment pollutant eco-friendly 7 / Versatile, quantitative Western blotting Measure high and low abundance proteins on the same blot ImageQuant Dynamic range Hyperfilm ECL Saturated bands Dynamic range pg pg LOD 9.8 pg DR 5000 pg 9.8 pg ( ) LOD 9.8 pg DR 310 pg 9.8 pg (10 1.5) ) Integrated Intensity (x10-5) Film (Exp: 5 min) IQ 350 (Exp: 6 min) transferrin (ng) 8 / 3 /

15 ImageQuant LAS 4000mini components Cooled CCD camera Down to -35 C ( standard -25 C ) Epi light connector Lens Lens connector Automatic filter changer Epi light Sample tray Tray rail Filter housing LAS-4000mini inside view 4 /

16 ImageQuant LAS 4000 mini Imaging applications Standard Upgrade 14 / 15 / August 13, /

17 ImageQuant LAS 4000 series components Cooled CCD camera Down to -35 C ( standard -25 C ) Epi light connector Lens Lens connector Automatic filter changer Epi light Sample tray Lifting table Transmitted light source connector Transmitted light source 16 / August 13, 2013 ImageQuant LAS 4000 series Imaging applications Standard 4000 & 4010 Standard 4010* versatility Upgrades, both systems * ImageQuant LAS 4000 can be upgraded to 4010 at any time 17 / August 13, 2013 Source: Fujifilm TM 6 /

18 Light sources and filter options versatility Excitation light source units Various filters 18 / August 13, 2013 What factors decide performance? Lens & aperture Chip size & pixel size CCD design Sensitivity vs. resolution Cooling & CCD calibration (dark flame) 19 / August 13, /

19 Calibration, correction and optimization Raw Dark Current CCD Calibration (dark frame) to remove background noise, which depends on temperature Corrected 20 / August 13, 2013 Large-aperture F0.85 lens Bright FUJINON F mm lens Specifically designed for the proprietary Super CCD chip by Fujifilm TM High sensitivity Designed for chemiluminescence Optional lens : Wide view lens (F mm (SIGMA)) 25 x 25 cm FUJINON VRF43LMD3 F0.85 LENS 21 / 8 /

20 CCD principle CCD: Charge Coupled Device 22 / CCD Size 2 Mega pixel, 13mm pixel 2 Mega pixel, 4.5mm pixel (31 per 6 inch wafer) (300 per 6 inch wafer) 24 / 9 /

21 Fujifilm TM Super CCD Pixel size (center to center) μm 2048 pixels 23.4 mm High pixel density gives better resolution than square matrices Binning up to 8 x 16 for high sensitivity 25 / CCD affording up to 6.3 megapixels Fujifilm took a different spin with the Bayer filter in its Super CCD sensor. Rather than a standard grid of RGB elements, Fujifilm turned everything at a 45 angle. This has two benefits: it makes the sensor elements larger (octagons rather than rectangles) and thus more sensitive, and the pixels are closer to each other than with a standard image sensor. Because of this, Super CCD sensors with 3 MP can produce 6 MP images nearly as good as those from true 6 MP sensors. 26 / 10 /

22 ImageQuant control software Intuitive and easy to learn Interactive image capture Optimal image settings every time 27 / / 11 /

23 Method/Tray position 29 / Tray position & size 30 / 12 /

24 31 / Focusing 32 / 13 /

25 Exposure 33 / Exposure type 34 / 14 /

26 38 / Sensitivity/Resolution 39 / 15 /

27 40 / After exposure Speed 44 / 16 /

28 Image data Speed save.gel.16 bit TIFF.8 bit tiff 46 / / 17 /

29 ImageQuant LAS 4000 ImageQuant control software IQTL analysis software 48 / ImageQuant TL analysis software 49 / 18 /

30 Live Demo! 52 / 19 /