User Manual Universal One
Contents
1. 2 Using DAB substrate supplied in kits WB R50 WB M50 and WB G50 e Take both 100ul of 100X DAB solution and 100ul of 100X D buffer and mix them with 10ml of distilled water Incubate this DAB mixture with washed the membrane at room temperature for a proper time period usually 3 10 minutes till desired color blots appear Wash the membrane with plenty of distilled water and then air dry it Re blot with different antibody Optional If needed the membrane after development can be reused for further staining with different primary antibodies Your valuable Western transferred membrane can be used efficiently with this protocol Tel 201 882 2593 Fax 201 882 2593 e Shake the membrane after Chemiluminescence developed not suitable after DAB developed with 10 ml of Stripping Buffer Cat 238 51 for 5 min at room temperature Recover the Stripping Buffer for reusing in future e Wash the membrane once with 10ml of PBS buffer and then rinse it with plenty of distilled water e Repeat above steps One Reaction or other procedure but using different primary antibody Wash and Development Vill Application Examples Detection of GAPDH proteins in cell lysate using One step procedure 1 hr comparing wit Regular WB procedure 4 hrs Primary Ab MAb ant GAPCOH Mires ten WE a 2 wa g a lt Fezular WE mt 1 2 3 4 GAPDH Primary Ab PAb anti GAPOH Upper left One step proce2. 10 tests 10X Rapid Wash Solution 100 ML Suitable for Goat Primary Antibodies 100X DAB Solution Set 2 ML ll Product Description IV Features Universal One Step Western Blot Kit patent pending is an innovative ande Easy a simple one step reaction a regular procedure needs 4 steps the most advanced Western blot kit With the kit your Western blot will be blocking 1st antibody binding washing and 2nd antibody binding no longer a time consuming and labor intensive procedure The procedure e Rapid the whole procedure takes less than one hour a regular immuno blot of a typical regular Western blot contains seven steps blocking washing takes 3 5 hrs primary antibody binding washing secondary antibody binding washing and developing and requires 4 5 hours In contrast the procedure of Universal One Step WB only contains one reacting washing and developing and requires as short as 60 minutes Currently we provide different types of Universal One step WB kit as shown on the above table Each kit is sufficient for detecting 10 without reuse to 40 if reused mini Universal suitable for most primary antibodies Sensitive comparable sensitivity with regular immuno blot Reusable antibodies and reagents can be reused Re stainable membrane can be re blotted with different antibodies Reproducible result is highly reproducible gel size membranes V Storage Ill Application for R amp D use
3. Procedure see below apparatus such as film cassettes and developer trays or auto film developing machine are also required iii Two step B Procedure e 1 BSA in PBS buffer optional0 Needed only when Two step 1 Enhance 1st Ab Soaking Add 10ml of Enhancer supplied in the kit Procedur B or Three step Procedure is selected see the procedure and Western transferred or dot blotted membrane to a proper container below for details gently shake the container to wet both sides of the membrane Note the membrane must be fully wetted by Enhancer before adding Ab add 5ug of Il Procedure primary antibody to the container mix well and continue shaking for 40 90 l i l l minutes longer incubation may needed if the specific affinity of primary Ab Universal One Step Western Blot Kits are designed for One step is weak or if less amount of primary Ab is used at room temperature Read Procedure but also compatible with Two step and Three step procedures Tips A above A proper procedure must be selected according to the quality of primary 2 Probe Incubation Rinse the membrane twice with plenty of distilled antibody to be used and the quality of result expected The following table water Mix 200ul of 50XWB Probe supplied in the kit with 10ml of 1 BSA provides a basic guide for selecting a proper procedure to obtain expected in PBS then add to the container continue shaking for 30 60 minutes at result room temperature 3 Wash Follow
4. WB M800 Load more protein s onto the SDS PAGE gel WB G800 WB R700 Universal One step IR Western Blot Kits WB M700 WB G700 For Infrared Detection Optimize transfer time and or the electrical WB R800 M700 current Make sure that there are no air bubbles between the membrane and gel IHC R60 IHC M60 IHC G60 IHC R61 IHC M61 IHC G61 E R70 E M70 E G70 Universal One step Indirect ELISA Kits E R71 E M71 E G71 with TMB or pNPP substrate Universal One step IHC Kits with DAB or BCIP NBT substrate Use high affinity and purified primary E R80 E M80 E G80 Universal One step Cell based ELISA Kits antibodies Select 2 step Procedure B or 3 step E R81 E M81 E G81 with TMB or pNPP substrate Increase the concentration of primary antibody E R90 E M90 E G90 Universal One step IgG Quantifctn ELISA or run an Antibody Titration to optimize primary l Kitsw TMB antibody concentration by One step Dot blot using serially diluted samples from 1 ug dot to 1 ng dot and serially diluted primary antibody Check the expiration date of the antibody as ell as the One step WB kit Use fresh or unexpired antibody and kit Increase antibody incubation time Especially hen the amount of antigen to be detected is very small and the affinity of primary antibody is weak increase incubation time to 2 hr or overnight Also if reagents stored in a refrigerator are used before being pre warmed to RT longer incubatio
5. of primary Ab is used at room temperature l oe 2 Wash Follow the Wash step under Common Procedure see below Tere lorment 3 Development Follow the Development step under Common Procedure see below Wash Wash Pree i t od Ab Tips A 1 The optimal amount 1 10ug 10ml in most cases of primary aoe Eindig Eindig antibody used in One step WB might be different from that used in a regular WB and is highly depending on its purity and affinity therefore to obtain best results a simple antibody titration using One step Dot Blot is recommended 2 The membrane must be fully covered by the solution VII Protocol during shaking 3 Extend incubation time for an additional 15 min if cold WB Enhancer is used before warming to room temperature 4 For convenience any step soaking or antibody binding can be left overnight at 4 C 5 If desired recover the used Reaction Mixture consisting of WB Enhancer primary antibody Probe and store at 2 8 C for up to 1 weeks or 20 C for 3 months or longer storage This One Reaction Mixture can be subsequently reused 3 4 times for testing the same antigen It may be required to add additional amount of primary antibody to compensate for the loss at each usage Regular WB Forgetit Additionally required materials not supplied in the kit e Primary antibody Purified high affinity IgG antibodies either polyclonal or monoclonal are preferred Although unpurified antibody o
6. Columbia Bio LLC www columbia bio com Universa SE E mail sales columbia bio com y n La TENA AAA One step Western Blot User Manual pr Universal One step Western Blot Kits For R amp D Use Only I Product Name Catalog Number amp Content Universal One step Western Blot Kit R i Shiels n ke 2 ML With SuperHi Chemiluminescence WB R51 Suitable for Rabbit Primary Antibodies Ortesis 10 Rapid vasi oom SuperHi Chemiluminescence 40ML Universal One step Western Blot Kit M Kit ee X He 2 ML With SuperHi Chemiluminescence WB M51 Suitable for Mouse Primary Antibodies TO ReiS 10 Rapid WASA Solution 100 ME SuperHi Chemiluminescence 40ML Universal One step Western Blot Kit G Kit ee 2 ML With SuperHi Chemiluminescence WB G51 Suitable for Goat Primary Antibodies ese 10 Rapid vvas Solution T04 MT SuperHi Chemiluminescence 40ML Universal One step Western Blot Kit R 7 aed ds 0 bi ne With DAB Substrate WB R50 i ODER 2 ML Suitable for Rabbit Primary Antibodies TO tests 10X Rapid Wash Solution 100 ML 100X DAB Solution Set 2 ML Universal One step Western Blot Kit M i o y 0 is hE With DAB Substrate WB M50 l 5 probe M0 2 MEJ Suitable for Mouse Primary Antibodies 110 tesis 107 Rapid Yvas t Solution 100 MI 100X DAB Solution Set 2 ML Universal One step Western Blot Kit G l Enhancer 100 ML With DAB Substrate WB G50 Kit 50XWB Probe G50 2 ML TES
7. dure 1 hr with rabbit anti GAPDH polyclonal antibodies Upper right Regular method 4 hrs using rabbit anti GAPDH polyclonal antibodies and goat anti rabbit 2nd antibody Lower left One step procedure 1 hr with mouse anti GAPDH monoclonal antibody Lower right Regular method 4 hrs using mouse anti GAPDH monoclonal antibody and rabbit anti mouse 2nd antibody Loaded cell numbers A549 cell lysate from lanes 1 to 6 are 10000 3000 1000 300 100 and 30 respectively Blots were developed with Chemiluminescence Il Rapid semi quantification of Flag tagged protein in purification fractions by One step Dot Blot Dilution Sup Ft W E1 E2 E3 E4 E5 E6 EF Std 1 0 a ebo didn 1 2 e 2000 1 8 667 ae 22 4 80 74 1 242 o 5 1 728 d g 1 2186 Serially diluted fractions middle dilution factors as indicated on left side containing Flag tagged protein P50 and purified P50 flag as standard right side were dot blotted onto an NC membrane 1ul dot and then detected by One step procedure using mouse anti flag from Sigma and Universal One step WB kit M Blots were developed with DAB Sup Supernatant before loading to column Ft Flow through W Wash fraction E1 E7 Eluted fractions 1 7 Std Standard purified P50 flag blotted amount as indicated on left side www columbia bio com IX Troubleshooting Guide xX Related Products Problem I Signal is weak or invisible Columbia Bio Catt WB R800
8. n time is needed ash time too long Reduce the wash time Problem Il High background or non specific bands on blot Select high affinity antigen specific primary antibodies Purified monoclonal or affinity purified polyclonal primary antibodies are preferred Increase Enhancer soaking time before adding B Probe and primary antibody Select 2 step or 3 step procedure Reduce the amount of primary antibody or the amount of WB Probe for One step Reaction Optimize the amount of antibody or WB Probe by dot blotting Non specific binding and cross reactivity of primary antibody Used too much primary antibody or Use clean equipments and freshly prepared buffers Wear gloves and use clean forceps to handle membranes Dilute the mixed substrate solution with 1 2 olume of distilled water Use chromogenic development reagents such as DAB which is less sensitive but produce lower background than Chemiluminescent reagent reagent too sensitive D Eiaa Tel 201 882 2593 Fax 201 882 2593 www columbia bio com
9. only e All reagents in Universal One Step Western Blot Kits can be stored at refrigerator 2 8 C except that WB probe and DAB solution must be stored Rapid Immuno blot Western blot and Dot blot for at 20 C Expiration date 6 months from the date of receipt if properly e Rapidly identifying a specific protein either purified or unpurified stored Rapid Wash Solution can be stored at room temperature for 1 year e Rapidly detecting a specific protein expressed in cells e Note Kit may be shipped in ambient temperature e Rapidly monitoring a target protein in a purification procedure just needs 1 2ul of sample e Rapidly semi quantifying a specific protein just needs 2u of sample e Rapidly screening or titrating antigen specific antibodies Tel 201 882 2593 Fax 201 882 2593 www columbia bio com VI Comparison of One step WB with Regular WB i One step Procedure 1 One Reaction Add 10ml of Enhancer supplied in the kit and Western transferred or dot blotted membrane to a proper container gently shake the container to wet both sides of the membrane Note the membrane must OnestepWB _ be fully wetted by Enhancer before adding Ab Mix Sug of primary So easy antibody with 200ul of 50XWB Probe supplied in the kit then add this mixture to the container mix well and continue shaking for 30 90 minutes p oe longer incubation may needed if the specific affinity of primary Ab is weak or if less amount
10. r serum is also suitable for the kits user may need to optimize the concentration of antibody or select a different procedure see Table of Procedure Guide below ii Two step A Procedure e Note All Kits are labeled with R M and G suitable for only Rabbit Mouse and Goat primary antibodies respectively User must pay attention to select right primary antibody to match different type kit 1 Enhancer soaking To a proper container add 10ml of Enhancer supplied in the kit and Western transferred or dot blotted membrane gently shake at room temperature for 30 60 minutes 2 Primary Ab Probe Incubation Mix 5ug of primary antibody with e Western blot Membrane Both Nitrocellulose and PVDF membrane are 200ul of 50XWB Probe supplied in the kit then add this mixture to the suitable for One step WB User must perform PAGE and Western above container mix well and continue shaking for 40 90 minutes longer transferring ahead of using this protocol incubation may needed if the specific affinity of primary Ab is weak or if less amount of primary Ab is used at room temperature Read Tips A above e X ray film amp its related reagents required only if Chemiluminescent 3 Wash Follow the Wash step under Common Procedure see below substrate is used Sensitive X ray films are preferred X ray film 4 Development Follow the Development step under Common developing related reagents excluding Chemiluminescence and
11. the Wash step under Common Procedure see below Table of Procedure Guide 4 Development Follow the Development step under Common 1 step 2 step A 2 step B 3 step Procedure see below Procedure Procedure Procedure Procedure a It Soakwith 20a iv Three step Procedure 1 Soak with Step s before Enhancer 1st Enhancer Enhancer 2 1st Ab ee wash for b 2 4stAb APB l 1 Enhancer Soaking Add 10ml of Enhancer supplied in the kit and development Probe incubate 2 Probe Western transferred or dot blotted membrane to a proper container gently shake at room temperature for 30 60 minutes 2 Primary Ab Incubation Add 5ug of primary antibody to the container mix well and continue shaking for 40 90 minutes longer incubation may Requirement Purified Purified high needed if the specific affinity of primary Ab is weak or if less amount of for aed Ab high affinity affinity primary Ab is used at room temperature Read Tips A above Light very 3 Probe Incubation Rinse the membrane twice with plenty of distilled Background Light low Light low eee R eae water Mix 200ul of 50XWB Probe supplied in the kit with 10ml of 1 BSA Moderate Modarate in PBS then add to the container continue shaking for 30 60 minutes at highest highest room temperature 4 Wash Follow the Wash step under Common Procedure see below 5 Development Follow the Development step under Common Procedure see belo
12. tic sheets Cover the membrane by gently pressing down the top cover sheet from one end to the other in order to eliminate bubbles and excessive substrate solution between the cover sheet and the membrane as showed in the following picture gt gt a gt WB Membrane Pre folded Side view Transparent sheet e In a dark room place an X ray film on the top of the covered membrane and expose for a proper time period usually 10 seconds to 5 minutes It is recommended to expose multiple X ray films for different time periods e Develop the X ray film in darkness or using an automatic developing machine Tips B 1 The pre folded transparent plastic sheet can be easily made by cutting a commercial Transparency Sleeve to a proper size with three open sides 2 If SaraWrap is used for covering avoid wrinkles occurring between the top sheet and WB membrane 3 SuperHi Chemiluminescence is a very sensitive substrate If developed X ray films are too dark try one or more of the following actions to reduce background 1 simply reduce the exposure time to as short as 1 second 2 extensively wash the membrane for another 5 min 3 expose X ray film with diluted substrate solution dilute the A amp B mixture 2 3 fold with distilled water but may need to increase exposure time 4 strip the blot with Stripping Buffer see Re blot below then re probe the membrane with a half amount of WB Probe or and a lower concentration of Primary Antibody
13. w Sensitivity High High Tel 201 882 2593 Fax 201 882 2593 www columbia bio com v Common Procedure 1 Wash e Prepare 1X Rapid Wash Solution Shake the bottle of 10X Rapid Wash solution as it might contain precipitate for 10 sec then dilute the solution to 10 times in volume with distilled water e Briefly rinse the membrane from above procedure with plenty of distilled water to remove free antibody and WB Probe e Wash the membrane for 3 5 minutes with 30 ml of 1X Rapid Wash Solution by aggressively shaking to remove non specific bound substances Rinse the membrane again with plenty of distilled water e Repeat once wash and rinse but twice if SuperHi chemiluminescence is used for development 2 Development 1 Using SuperHi Chemiluminescence supplied in kits WB R51 WB M51 and WB G51 Note Universal One step WB kits are compatible with most chemiluminescent substrates but detection sensitivity may vary e Mix both 2 ml Solution A and 2 ml Solution B of chemiluminescent substrate in a proper container e Drain excessive water on the surfaces of the washed membrane by touching one end on a clean tissue paper Wet the membrane with the mixed substrate solution the protein side of the membrane must be fully wetted by the solution e Drain excessive liquid on the membrane by touching one end of the membrane on a clean tissue paper Place the membrane protein side up between pre folded transparent plas
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