VisPROTM 5 minutes Protein Stain Kit
Contents
1. or nylon membrane for Western blotting The stained gel is also re stainable by VisPRO 5 minutes Protein Stain and all other known staining methods such as Coomassie brilliant blue stain silver stain and Sypro Ruby stain Switch NOW to VisPRO 5 minutes Protein Stain and See how it changes your researches Product component VisPRO 5 minutes Protein Stain Kit 1 nanogram scale VP 1005 500 Solution 5X Sensitizing Solution VP 1005 1 500 500ml 1 bottle Solution Il 5X Developing Solution VP 1005 2 500 500ml 1 bottle User s manual 1 booklet VisPRO 5 minutes Protein Stain Kit 1 nanogram scale VP 1005 125 Solution 5X Sensitizing Solution VP 1005 1 125 125ml 1 bottle Solution II 5X Developing Solution VP 1005 2 125 125ml 1 bottle User s manual 1 booklet Please 5X dilute with ddH2O before using Safety Information Please wear gloves lab coat and goggles while operating Prevent contact product directly In case of contacting wash with large amount of water Storage VisPRO 5 minutes Protein Stain Kit can be stored at room temperature Please use up the opened product in 2 years Rock the bottle gently before using A Quick Staining Protocol 1 Dilute 1 volume of 5X VisPRO with 4 volume of ddH O before using 2 After electrophoresis move the ge into staining box with dark background Black staining box is available BS10017BS1002 3 Add Solution to immerse the gel Generally 22. 0 mL is sufficient for one 8 x 10 cm gel Gently agitate the staining box 50 75 rpm for 5 minutes 4 Discard Solution Wash the gel briefly with ddH O for 5 seconds 5 Add Solution II directly into the staining box Solution II SHOULD NOT be poured directly onto the gel surface It will cause uneven background Vigorously agitate the staining box by hand The background of gel should turn white and the protein bands or spots should be visualized in less than 20 seconds 6 Discard Solution II immediately Wash the gel twice with ddH O T Store the gel in ddH O Depending on the quality of distilled water the stained image can be sustained without protein diffusion from weeks to months For better storage stained gel can be sandwiched between two transparency sheets and ziplocked in a plastic bag at 4 C In this way gel images can be clearly discerned after months It is possible to perform manual spot picking from the stored gels Switch NOW to VisPRO 5 minutes Protein Stain and See how it changes your researches B Highly Sensitive Staining Protocol 1 Dilute 1 volume of 5X VisPRO with 4 volume of ddH O before using 2 After electrophoresis move the gel into staining box with dark background Black staining box is available BS1001 BS1002 3 Add SDS electrophoresis buffer Tris glycine buffer 100 ml for one 8 X 10 cm gel Gently agitate the staining box 50 75 rpm for 5 minutes 4 Discard SDS electrop
3. VISUA PROTE L OTEIN VP1005 500 VP1005 125 VisPRO 5 minutes Protein Stain Kit 5X concentrate 1 nanogram grade Visual Protein BiotechnologyCorp Innovation amp Incubation Center No 510 ZhongZheng Rd XinZhuang City Taipei County 24299 Taiwan P O Box 1 239 XinZhuang Taipei County 24299 Taiwan www visualprotein com Tel 886 2 8660 6212 Fax 886 2 8660 2947 Introduction VisPRO 5 minutes Protein Stain Kit 1 nanogram grade provides a quick and easy way to read your protein gel No fixation is required Simply add the pre made solution to your gel and find the protein bands or spots in less than 5 minutes VisPRO 5 minutes Protein Stain Kit applies the principle of zinc stain a negative stain method The formation of zinc imidazole complex on polyacrylamide turns the gel to white while the protein area prevents the stain and remains transparent VisPRO 5 minutes Protein Stain Kit perfectly matches to current proteomic research Generally more protein spots can be detected in 2 DE gels by this robust staining method than silver and Sypro Ruby stain Moreover the unfixing nature has it very ideal for subsequent analysis by mass spectrometry Proteins developed by VisPRO 5 minutes Protein Stain usually have higher rate of good annotation VisPRO 5 minutes Protein Stain Kit is also compatible to other techniques The stained gel can be electro eluted for recovering proteins or electro transferred onto PVDF
4. ash the tuned gel before adding Solution I 20 mL 50 mL and 100 mL of Solution I II are sufficient to stain 8x10cm 16x18 cm and 24x20 cm 2 DE gels respectively Wy ally A lt r Because the 2 DE gels is often thicker it is recommended to extend incubation of Solution I to 10 15 minutes for better staining results It is recommended to cut the desired protein spots for the stained gel right after the staining C Image acquisition 1 Scan the stained gel in the flatbed scanner with transparency unit at transparent mode Turn on the option of reversing image recommend instrument 2 When using CCD camera to document the gel image a black background has to be place under the gel However the obtained gel image generally has less contrast D Destaining and Restaining I Destaining by SDS electrophoresis buffer regular Tris Glycine buffer with SDS 1 The stained gel can be destained by SDS electrophoresis buffer in minutes 2 f restaining of gel is required just following the staining procedure starting from Solution I ll Destaining by 10 acetic acid 1 The stained gel can also be destained by 10 acetic acid in less than 1 minute Fixation of protein will occur at the same time 30 minutes fixation should be sufficient for complete immobilization of proteins 2 If restaining of the acetic acid fixed gel is required wash the gel three times by distilled water for 10 minutes Equi
5. ay Juang Ai Ling Hour Pei Sing Chen Hui Ming Huang Szu Yu Wu Jen Chieh Lee Tzung Lin Tsai and Han Min Chen PROTEOMICS 2009 9 696 709 3 Yi Jen Liau Lisa Wen Jei Fu Shawc Chi Tsai Lin Journal of Biotechnology 2007 131 84 91 4 Shih Chung Chen Bin Huang Yu Chi Liu Kou Gi Shyu Pen Y Lin and Danny Ling Wang Biochemi cal and Biophysical Research Communications 2008 377 1274 1278 5 Ching Yu Lin Hui Ming Huang and Han Min Chen BIOTECHNIQUES 2006 41 560 564 Appendex B Comparison of staining methods VisPRO 5 minutes Methods Protein Stain Sypro Ruby Silver Stain CBR Stain Preparation of f f solutions 0 min 5 min 20 min 0 min Fixing Step O min 1 hr 1hr overnight 30 min Stain 5 min overnight 30 min overnight 3 hr Image Dispaly 30 sec 30 min 30 min overnight Total Time 5 min 30 sec 3 18 hr 4 20 hr 1hr 8hr Imaging gt UV 302 nm R E Visble light or 480 620 nm Visble light Visble light Irratated or n Acetic acid gt Eo Acetic acid gt i i Methanol 4 Methanol toxic chemical ethano Gimeraen de ethano Sensitivity lt 1 ng 1 ng 1 ng 50 ng eee 1 200 ng 1 1000 ng 4 80ng 50 500 ng including operating gt developing and destaining time Quantitative might vary depending on the nature of proteins FAQs about VisPRO 5 minutes Protein Stain Kit 1 10 11 12 How can digitalize VisPRO stained gel images What instruments are needed Ans Option 1 We suggest using a f
6. horesis buffer 5 Add Solution to immerse the gel Generally 20 ml is sufficient for one 8 x 10 cm gel Gently agitate the staining box 50 75 rpm for 5 minutes 6 Discard Solution Wash the gel briefly with ddH O for 5 seconds 7 Add Solution II directly into the staining box Solution Il SHOULD NOT be poured directly onto the gel surface It will cause uneven background Vigorously agitate the staining box by hand The background of gel should turn white and the protein bands or spots should be visualized in less than 20 seconds DO NOT develop more than 1 Minutes or it will cause overstaining 8 Discard Solution II immediately Wash the gel twice with ddH O 9 Store the gel in ddH O Depending on the quality of distilled water the stained image can be sustained without protein diffusion from weeks to months For better storage stained gel can be sandwiched between two transparency sheets and ziplocked in a plastic bag at 4 C In this way gel images can be clearly discerned after months It is possible to perform manual spot picking from the stored gels 9 Important notification for staining 2 DE gels In some circumstance the pH value and wetness of 2 DE gels are greatly changed during long time of electrophoresis It is highly recommended to tuning your 2 DE gel conditions by submerge it in SDS electrophoresis buffer Tris glycine buffer for 5 10 minutes before adding Solution It is not required to w
7. hould be iS poor restricted to about 20 30 seconds Background is not white and the contrast iS poor The pH of the original gel might be drifted too acidic The original gel is too dry Tuning your gel in SDS electrophoresis buffer before staining 1 Pour Solution II into area of staining boxes without gels 2 Add Solution II quickly 3 Agitation vigorously when Solution II is added 4 Wear gloves Clean all glassware and eletropho retic apparatus Solution Il is poured directly onto gel surface Uneven background The addition of Solution Il is too slow The agitation of Solution Il is not vigorous enough The contamination of protein from environments 9 Please note All unsatisfactory stained gel can be destained by SDS electropho resis buffer and restained again Accessories NeneneneeanansenenenenseaeeeeHeneHenSeaHAeeHAOSHSHSEAHAOSENAOENSHSEAESOSEHOOSHSHSESESOSEHOGEHE OREOOEESHOHENORSESOOESSHOHENSRSEEOOESSHONSNSUSEEOOESSOSHENSUSEEOOESSOSHENSUSEEOOESHSSHSHORSEEOSI OREHEORENESEKONONEHEOOEEOOEKENSEHSOOEEOOEAONSAEHSOSEEOSAONSEHEOSEESOOKONSQEHEOOENESERENOR SHEE GL1001 BS1001 BS1002 Gel Lighting Plate Black Staining Box Small Black Staining Box Large Size 297L 210W mm Size 125L 95W 35H mm Size 175L 175W 42H mm Appendex A 1 Fernandez Patron C Castellanos Serra L Rodriguez P BIOTECHNIQUES 1992 12 564 573 2 Ching Yu Lin Vinchi Wang Hao Ai Shui Rong Hu
8. latbed scanner with transparency unit to document the reverse stained gel images While scanning turn on the transparency mode and save the image into 8 or 16 bit grayscale TIF file For optimal image quality stretching the level of image to maximal 0 255 is suggested before saving the files Option 2 Gels can also be documented by flatbed scanner without transparency unit However a black background should be placed behind the gel while scanning Note Using a CCD camera to capture gel images is not recommended Central light spots on the documented images are generally observed from the reflection of epi light source How can I store the stained gels Ans Option 1 For long term storage stained gel can be sandwiched between two transpar ency sheets and ziplocked in a plastic bag at 4 C In this way gel images can be clearly discerned after months It is possible to perform manual spot picking from the stored gels Option 2 Stored in solutions like distilled water is not recommended However the reverse stained gel can be preserved in distilled water for weeks Sometimes fading of gel image may be observed for laboratories equipping with distilled water of pH lower than pH 7 Switching the storage solution to 100 mM Tris buffer at pH 7 2 might remedy above fading phenomena How can I see stained gels more clearly when I pick protein spots in gel Ans We recommend using the Gel Lighting Plate Product code GL1001 to vis
9. librate the gel in SDS electrophoresis buffer 5 minutes before restaining E Visualize your gel 1 The location of protein on the stained gel can be easily visualized by eyes under black background such as in the black staining box BS1001 BS1002 2 When spot picking is desired it is recommended to use gel light plate GL1001 for best visual effect Other downstream applications A Mass spectrometry 1 Move the cutting protein spots or bands into micro centrifuge tube Eppendorf brand is recommended 2 Destained and fix the gels in 10 acetic acid for 30 minutes 3 Wash the fixed gel thoroughly with three changes of ddH O 4 The washed gel can then be stored at 2 C or processed directly according to the standard mass sample preparation procedure B Electroelution Electroblotting 1 Before electroelution or electroblotting is performed Wash the gel 10 minutes by Tris glycine buffer without SDS 2 Perform electroelution or electrobltting according to standard procedures C Restaining by other staining methods 1 Fix the gel by 10 acetic acid for 10 minutes before performing Coomassie brilliant blue stain silver stain Sypro Ruby stain or other staining method 2 Destain the gel by Tris glycine buffer without SDS if activity stain is required Trouble shooting Possible cause Remedy Background is too The gel may be over The optimal development by white and the contrast developed Solution IlI s
10. resent uneven and mist like background after staining Is it remediable Ans It might happen if pouring Solution II directly onto gel surfaces or agitating is not vigorous enough while development You can destain the gel and then restain it accord ing to the manual s instructions Can stain the acidic fixed gels by VisPRO 5 minutes Protein Stain Kit Ans Definitely You should wash the fixed gel thoroughly by distilled water of 10 minutes for three times and then equilibrate the gels in SDS electrophoresis buffer for another 10 minutes The washed gels can be subsequently developed by VisPRO 5 minutes Protein Stain Kit following the standard staining protocol Why I can t re stain the acidic fixed gel as claimed Ans Insufficient washing may result in an acidic environment residing in the gels Just performing another thorough washing by distilled water and equilibrating by SDS electro phoresis buffer may remedy Can use VisPRO 5 minutes Protein Stain Kit for gels from electrophoresis conditions other than Tris Glycine SDS system Ans Definitely However you should wash such gels by distilled water for 10 minutes and equilibrate in SDS electrophoresis buffer for another 10 minutes The washed gels can be subsequently developed by VisPRO 5 minutes Protein Stain Kit following the standard staining protocol What is the dynamic range of staining for VisPRO 5 minutes Protein Stain Kit Ans Generally the d
11. ualize the gel It has been proved that gel lighting plate is excellent for direct observing the reverse stained gels Reference Ching Yu Lin Hui Ming Huang and Han Min Chen Use of backlit light plate to enhance visualization of imidazole zinc reverse stained gels BioTechniques 41 560 564 November 2006 Why the sensitivity of stain doest not reach to 1 ng of protein as claimed Ans The gel is over stained VisPRO 5 minutes Protein Stain is a negative staining method The gel background is the target proteins are not the staining targets Over staining will decrease the sensitivity The optimal development time by Solution II limits to 20 30 seconds It should not exceed 60 seconds Why the background of my gel did not turn white within 30 seconds Ans The pH is drifted VisPRO 5 minutes Protein Stain is highly sensitive to the changing of environment pH At acidic environment it takes longer than 30 seconds for turning the background of gel to white Either the electrophoretic gels become acidic due to prolonged electrophoretic procedure or the mild acidic distilled water may result in the DH drifting to acidic Note We recommend to soak the electrophoretic gels into the SDS electrophoresis buffer containing glycine tris and SDS pH 8 4 for 10 minutes before performing VisPRO 5 minutes Protein Stain Generally employing such procedure all gels can be developed within 30 seconds with excellent results Why does gel p
12. ynamic range of staining for VisPRO 5 minutes Protein Stain Kit is from 0 to 200ng which is better than silver stain lt 100 ng How should prepare the VisPRO stained gel for downstream analysis of mass spectrometry MS Ans 1 Move the cutting protein spots or bands into micro centrifuge tube Eppendorf brand is recommended 2 Destain and fix the gels in 10 acetic acid for 30 minutes 3 Wash the fixed gel thoroughly with three changes of ddH20O 4 The washed gel can then be stored at 20 degree C or processed directly according to the standard mass sample preparation procedure How can destain the reverse stained gels Ans Option 1 Destaining by SDS electrophoresis buffer regular Tris Glycine buffer with SDS for 10 minutes This destaining method is used where restaining by VisPRO 5 minutes Protein Stain or membrane transferring is required Option 2 Destaining by 10 acetic acid for 5 30 minutes This destaining method is used where fixing of the protein is required such as preparing the samples for mass spectrometry MS or restaining by other gel staining methods such as silver stain CBB stain or Sypro Ruby stain
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