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Western manual_YZY_06242010.ai

1. M Tag Protein G Ab L Protein A 50 ng 50 ng Protein G 50 ng 50 ng Mouse Ab 2 ug 2 ug M Tag 20 ng 8ng 20 ng 8ng Figure 2 Western blot detection of multiple tag fusion protein by both classical western and ONE HOUR IP Western using kit L00232 Both blots are developed using Mouse Anti Trx tag Monoclonal Antibody GenScript A00180 and the LumiSensor Chemiluminescent HRP Substrate that is included in kit L00232 lt 13 ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR IP Western Kits continued Examples continued 3 Comparison of ONE HOUR IP Western blot with classical western blot using goat primary antibody ONE HOUR Classical Western IP Western 123 4 123 4 Ab H M Tag Protein G Protein A 50 ng 50 ng Protein G 50 ng 50 ng Goat Ab 2 ug 2 ug M Tag 20 ng 8ng 20 ng 8ng Figure 3 Western blots for the detection of multiple tag fusion protein by both classical Western and ONE HOUR IP Western using kit L00233 Both blots are developed using goat antibody anti HA GenScript A00168 and the LumiSensor Chemiluminescent HRP Substrate included in kit L00233 14 ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western Cat No L00397 Fluorescent Kit Reagents Needed This procedure is optimized for a sheet of 7 5 X 8 0 cm membrane but reagent volumes can be sc
2. Reduce the exposure time If both the signal and background are high wait for a few minutes before exposing the film 29D ONE HOUR Western Detection System www genscript com Troubleshooting ONE HOUR Fluorescent Kit Problem Probable Cause Solution The signal is Too little protein is loaded weak or invisible Load more protein s onto the SDS PAGE gel There is poor transfer efficiency Optimize the transfer time and or the electrical current Make sure that there are no air bubbles between the membrane and the gel The primary antibody has a low affinity for the antigen Increase the incubation time of the membrane in WB 2 containing Mixture 1 Increasing antibody concentration can also improve signal The primary antibody has a low affinity for the antigen Reducing wash time can increase the signal for low affinity antibody Instead of washing for 10 min x 3 wash for 5 min x 3 to increase signal There is high Too much primary antibody was used background Reduce the amount of primary antibody and reduce WB 1 accordingly The primary antibody has non specific binding or cross reactivity with the blocking reagent Use an alternate Pretreat A b M01057 The wash time is too short Adding additional washing steps can further decrease background The equipment or reagents have become contaminated Use a clean con
3. ONE HOUR Western Detection System Update Date June 25 2010 User Manual GenScript The Biology CRO ONE HOUR Western Detection System www genscript com Table of Contents Kit Contents 22 022 r nnn rrr nrc nr nnn rnc ete 1 Introduction Ae a sorter costae aostiennn sine sori cinitinnnin ise minim tinicinrinieininin remininiaiminitinin min 3 Quick Selection Guide 2 0 2200220 rrr rrr nnn ner c cr neee 4 Protocols lt s22s22ersnss5 sete erareeech esse E a A a 5 ONE HOUR Western Basic Standard Advanced Kits 5 ONE HOUR IP Westem Kits 222 lt s 2424e eees eeedesce seen ed tede esas seek eee 10 ONE HOUR Westem Fluorescent Kit sie 16 0 she nusdeoedde nee eae eee nA eE Dt 15 ONE HOUR Western Multiplex Fluorescent Kit 19 Troubleshooting 2222 seco o e e EERE EEE 21 Technical Support 220002002 nnn errr nnn errr 25 Patent Pending 2 2 2222 2 222 eee ee eee e ence eee eee eres 26 ONE HOUR Western Detection System www genscript com Kit Contents Type of products Product Cat No ONE HOUR Western Basic Kit Rabbit L00204 ONE HOUR Western Basic Kit Mouse L00205 ONE HOUR Western Basic Kit Goat L00399 ONE HOUR Western S
4. ONE HOUR IP Western Kits Problem Probable Cause Solution The signal is Too little protein is loaded Load more protein s onto the weak or invisible SDS PAGE gel There is poor transfer efficiency Optimize the transfer time and or the electrical current Make sure that there are no air bubbles between the membrane and the gel There is high There is non specific binding background cross reactivity of primary antibody The blot shows protein A G or A G carryover contamination Change antibodies Use a highly specific primary antibody Affinity purified primary antibodies are preferred Increase the Protein A G blocking time to ten minutes or longer Add some Protein A amp G blocker to the IP WB 3 solution Instead of 100X try 200X The heavy chain or light chain of the antibody is still visible There is too much primary antibody If using the rabbit kit use more protein G blocker Load less sample to reduce antibody loading Use the same amount of primary antibody but less WB 1 solution For example mix 10 ug of primary antibody with 80 ul of WB 1 solution Reduce both the volume of the WB 1 solution and the amount of primary antibody added to it in step 1 while keeping the proportions the same For example instead of using 100 ul of WB 1 with 10 ug or more of primary antibody use 50 ul of WB 1 solution with 5 ug of primary antibody The signal development time is too long
5. electrical current Make sure that there are no air bubbles between the membrane and the gel The primary antibody has a low affinity for the antigen Increase the incubation time of the membrane in WB 2 containing Mixture 1 Increasing antibody concentration can also improve signal The primary antibody has a low affinity for the antigen Reducing wash time can increase the signal for low affinity antibody Instead of washing for 10 min x 3 wash for 5 min x 3 to increase signal There is high background Too much primary antibody was used Reduce the amount of primary antibody and reduce WB 1 accordingly The primary antibody has non specific binding or cross reactivity with the blocking reagent Use pretreat A b M01052 Customers can also use the Quick Block Optimization Kit to find the best blocking reagent The wash time is too short Adding additional washing steps can further decrease background The signal development time is too long The equipment or reagents have become contaminated Reduce the exposure time If both the signal and background are high wait for a few minutes for background signal to go down before exposing the film Use a clean container for each rinse and wash step Wear gloves and use clean forceps to handle membranes 294 ONE HOUR Western Detection System www genscript com Troubleshooting continued
6. 6 and 7 respectively ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western Basic Standard Advanced Kits continued Examples continued Comparison of the two ONE HOUR Western Kits of different sensitivities using polyclonal antibodies Two similar blots were processed with the same procedures using different ONE HOUR Western Kits Standard L00204C and Advanced L00241 10 ug and 2 5 ug of Rabbit Anti GST tag Polyclonal Antibody GenScript A00097 respectively were used with the two kits to detect GST protein The results are shown in Figure 2 Standard Kit 1 2 3 4 5 6 eee 25 0 125 6 25 3 10 1 50 0 74 Advanced Kit 1 2 3 4 5 6 as _ lt GST Loading 25 0 12 5 625 3 10 1 50 0 74 GST ng Figure 2 Western blots for the detection of GST protein using different ONE HOUR Western Kits Standard L00204C and Advanced L00241 25 0 12 5 6 25 3 10 1 50 and 0 74 ng of GST protein were loaded onto Lane 1 2 3 4 5 and 6 respectively ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR IP Western Kits Cat No L00231 L00232 L00233 Reagents Needed 1 This procedure is optimized for a sheet of 7 5 x 8 0 cm membrane The reagent volumes can be scaled up or down according to the size of the membrane used 2 The product is optimized to block up to 2 ug of antibody per lane Do not load more t
7. Kit Fluorescent Kits Pretreat Solution A 100 ml 100 ml Pretreat Solution B 100 ml 100 ml WB 1 Solution 2 ml 2 ml WB 2 Solution 100 ml WB M Solution 100 ml 10X Wash Solution 125 ml 125 ml User Manual 1 1 ONE HOUR IP Western Kits Components L00231 Rabbit L00232 Mouse L00233 Goat Pretreat Solution A 50 ml 50 ml 50 ml Pretreat Solution B 50 ml 50 ml 50 ml Protein A amp G blocker 100X 0 5 ml 0 5 ml Protein G blocker 100X 0 5 ml IP WB 1 solution 0 5 ml 0 5 ml 0 5 ml IP WB 2 solution 0 5 ml 0 5 ml 0 5 ml IP WB 3 solution 50 ml 50 ml 50 ml 5X Wash solution 125 ml 125 ml 125 ml WestClear Nitrocellulose 5 Sheets 5 Sheets 5 Sheets Membrane 0 2 um 7 5 x 8 cm LumiSensor Chemiluminescent 2x75 ml 2x 7 5 ml 2x 7 5 ml HRP Substrate User Manual 1 1 1 ONE HOUR Component L00276 QuickBlock Kit Pretreat Solution A 2 x 50 ml Pretreat Solution B 2 x 50ml User Manual 1 ONE HOUR Western Detection System www genscript com Introduction Storage The ONE HOUR Western Detection System is designed to produce a high signal with a low background for quick and clear western analysis of proteins GenScript s breakthrough ONE HOUR Western technology simplifies the classical western blot analysis by skipping the secondary antibody binding and washing steps The kits reduce the total western blot analysis time from 4 5 hours down to only one hour Easy to perform Quick and simple procedure High
8. sensitivity The sensitivity of the ONE HOUR Western is comparable to or better than that of the classical 4 5 hour procedure depending on the quality and quantity of antibodies used Highly reproducible results e Less optimization needed than the classical method e Secondary antibody is included e No special labeling required for primary antibody GenScript Classical ONE HOUR Western Western Blot Detection System Detection IU g Zz ile Z 4 lag MY Blocking Pretreatment ere Primary Antibody Loa 4i ONE HOUR Western Kit Primary Development Antibody Secondary Antibody Development lt a 4 5 hours Store WestClear Nitrocellulose Membrane at room temperature Store the rest of the kit at 4 C It will remain stable for six months Do not freeze the kit or any of its components ONE HOUR Western Detection System www genscript com Quick Selection Guide ONE HOUR Western Detection Kits ENET 6 pianga sea a HRP ETS ie ee si ae rep a anne Basic Kits aN caries Advanced Standard Kits Rabbit Mouse Goat Rabbit Mouse Goat L00241 L00242 L00243 L00204 L00205 L00399 Low background No dark room or film is needed Standard Standard Kits with TMB Rabbit Mouse Goat Rabbit Mouse Goat L00204C L00205C L00228 L00204T L00205T L00228T Sensitivity Potting a a ONE HOUR Western Fluorescent Kits Any primary antibody can
9. www genscript com Protocols continued ONE HOUR IP Western Kits continued Prepare Mixture 1 Before or during protein transfer prepare Mixture 1 by mixing 100 ul of IP WB 1 with 10 ug or more of the primary antibody in a microcentrifuge tube Vortex Mixture 1 for a few seconds and spin down briefly to collect the solution in the bottom of the tube Incubate Mixture 1 at room temperature for at least 40 minutes Longer incubation is preferred For overnight incubation store Mixture 1 at 4 C Note If less than 10 ug of primary antibody is to be used in Western blot the volume of IP WB 1 should be reduced accordingly For example mix 50 ul of IP WB 1 with 5 ug of primary antibody to make Mixture 1 The other reagents do not need to be adjusted Pretreatment of Membrane and Preparing Mixture 2 Mix 10 ml of Pretreat Solution A with 10 ml of Pretreat Solution B in a plastic container to make the pretreat solution mixture Incubate the membrane after protein transfer into the pretreat solution mixture on a shaker for five minutes at room temperature After incubation rinse the membrane twice with 15 ml of 1X wash solution Meanwhile prepare Mixture 2 by adding 100 ul of IP WB 2 to Mixture 1 Vortex Mixture 2 for a few seconds and spin down briefly to collect the solution in the bottom of the tube Incubate Mixture 2 at room temperature for five minutes Optional Protein A and Protein G Active Site Blocki
10. 2 Sigma T6074 B Actin THE Anti B actin 6 ug IRDye 800CW Goat Anti Mouse 3 ug Monoclonal Antibody Mouse LI COR 926 32210 GenScript A00702 GAPDH Goat Anti GAPDH 4 ug IRDye 680 Donkey Anti Goat 2 ug Polyclonal Antibody LI COR 926 32224 GenScript A00191 GST Rabbit Anti GST 6 ug IRDye 800CW Goat Anti Rabbit 3 ug Polyclonal Antibody LI COR 926 32211 GenScript A00097 A B Cc 1234 67 8 ge Oe a a ee A ey eS a Tubulin gt 8 Actin GAPDH NGST M 5 02 5 1 2 0 60 3 0 16 0 M 5 02 5 1 20 6 0 3 0 16 0 M 5 02 5 1 2 0 60 3 0 16 0 Hota Coll Lysate pg O 081 6 3 16 2 12 550 O 0 81 63 16 2 12 5 50 O 0 81 6 3 16 2 12 5 50 GST ng Figure 1 Multiplex Fluorescent Western blots for the detection of a Tubulin B Actin GAPDH and GST proteins using the ONE HOUR Western Multiplex Fluorescent Kit L00398 A 700 nm fluorescence image B 800 nm fluorescence image C The two fluorescence colors were imaged simultaneously in a single scan on a LI COR Odyssey Infrared Imaging Systems M is the Protein Marker for Fluorescent Western GenScript M00124 20 ONE HOUR Western Detection System www genscript com Troubleshooting ONE HOUR Western Basic Standard Advanced Kits Problem Probable Cause Solution The signal is Too little protein is loaded Load more protein s onto the weak or invisible SDS PAGE gel There is poor transfer efficiency Optimize the transfer time and or the
11. Cat No L00398 Multiplex Fluorescent Kit Reagents Needed This procedure is optimized for a sheet of 7 5 X 8 0 cm membrane but reagent volumes can be scaled according to the size of the membrane used Reagents not provided 1 Purified primary antibodies Affinity purified antibodies are recommended 2 Fluorescent dye labeled secondary antibodies Several vendors provide these kinds of antibodies LI COR and Rockland provide IRDye 680 800 labeled secondary antibodies Pierce provides DyLight 680 800 labeled secondary antibodies Invitrogen provides Alexa Fluor 680 labeled secondary antibodies Before use prepare the following 1X wash solution Dilute 12 5 ml of 10X wash solution with 112 5 ml of distilled or filtered water to make 125 ml of 1X wash solution If any precipitate forms in the 10X wash solution during storage incubate the bottle in a warm or hot water bath up to 50 C with occasional mixing until all the precipitate disappears Use 15 ml of 1X wash solution for each rinse and 20 ml of 1X wash solution for each wash Prepare Mixture 1 Before or during protein transfer prepare Mixture 1 by mixing the primary antibody and fluorescent dye labeled secondary antibody in WB 1 For multiple primary antibodies multiple Mixture 1 s need to be prepared separately in different tubes For each primary antibody add 2 10 ug of the antibody to 50 ul of WB 1 in a microcentrifuge tube then add 1 5 ug of t
12. aled according to the size of the membrane used Reagents not provided 1 Purified primary antibodies Affinity purified antibodies are recommended 2 Fluorescent dye labeled secondary antibodies Several vendors provide these kinds of antibodies LI COR and Rockland provide IRDye 680 800 labeled secondary antibodies Pierce provides DyLight 680 800 labeled secondary antibodies Invitrogen provides Alexa Fluor 680 labeled secondary antibodies Before use prepare the following 1X wash solution Dilute 12 5 ml of 10X wash solution with 112 5 ml of distilled or filtered water to make 125 ml of 1X wash solution If any precipitate forms in the 10X wash solution during storage incubate the bottle in a warm or hot water bath up to 50 C with occasional mixing until all the precipitate disappears Use 15 ml of 1X wash solution for each rinse and 20 ml of 1X wash solution for each wash Prepare Mixture 1 Before or during protein transfer prepare Mixture 1 by mixing primary antibody and fluorescent dye labeled secondary antibody in WB 1 Add 2 10 ug of primary antibody to 100 ul of WB 1 in a microcentrifuge tube then add 1 5 ug of fluorescent dye labeled secondary antibody the amount of secondary antibody is 50 of the primary antibody used to the same tube Vortex Mixture 1 gently for a few seconds and centrifuge briefly Incubate Mixture 1 in the dark at room temperature for at least 40 minutes Refer to manufa
13. be used Western analysis for multiple proteins Fluorescent Kit L00397 Multiplex Fluorescent Kit L00398 ONE HOUR IP Western Kits eee from protein A G IP Kits l l Rabbit Mouse Goat L00231 L00232 L00233 Note Customers need to provide both the primary and secondary antibody 4 ONE HOUR Western Detection System www genscript com Protocols ONE HOUR Western Basic Standard Advanced Kits Cat No L00204 L00205 L00399 L00204C L00205C L00228 L00204T L00205T L00228T L00241 L00242 L00243 Reagents Needed This procedure is optimized for a sheet of 7 5 x 8 0 cm membrane However reagent volumes can be scaled up or down according to the size of the membrane used Reagents not provided Purified primary antibodies Affinity purified antibodies are recommended Further optimization may be needed if the serum containing the antibody is to be used Before use prepare the following 1X wash solution Dilute 25 ml of 5X wash solution with 100 ml of distilled or filtered water to make 125 ml of 1X wash solution If any precipitate forms in the 5X wash solution during storage incubate the bottle in a warm or hot water bath up to 50 C with occasional mixing until all the precipitate disappears Use 15 ml of 1X wash solution for each rinse and 20 ml of 1X wash solution for each wash Prepare Mixture 1 Before or during protein transfer prepare Mixture 1 by mixi
14. cturer s recommendations of appropriate amounts of antibody 15 ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western Fluorescent Kit continued Pretreat Membrane Just before the protein transfer from gel to membrane is complete mix 10 ml of Pretreat Solution A with 10 ml of Pretreat Solution B in a plastic container Western blot box GenScript M00100 to make the pretreat solution mixture Always prepare and use a fresh solution mixture Place the membrane directly in the pretreat solution mixture and incubate on a shaker for five minutes at room temperature After incubation rinse the membrane twice with 15 ml of 1X wash solution Final Incubation of Pretreated Membrane a Add Mixture 1 to 10 ml of WB 2 in a Western blot box GenScript Western Blot Box Black M00103 and mix well Incubate the membrane in this solution WB 2 containing mixture 1 on a shaker at room temperature for 40 minutes Protect this box or bag from light during incubation This solution WB 2 containing mixture 1 may be recovered and reused up to three times if stored at 4 C However this may cause variations to arise due to changes in antibody concentration and carryover contamination b Rinse the membrane once with 15 ml of 1X wash solution Wash the membrane on a shaker three times for ten minutes each with 20 ml of 1X wash solution Protect box or bag from light during wash U
15. de using this product or its components for any commercial purposes For commercial use please contact GenScript at info genscript com 296 GenScript The Biology CRO GenScript USA Inc 860 Centennial Ave Piscataway NJ 08854 Tel 732 885 9188 732 885 9688 Fax 732 210 0262 732 885 5878 Email product genscript com
16. en protected from light the working solution A B remains stable for several hours at room temperature Summary of Working Solution Preparation 0 05 ml is needed per cm of membrane ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western Basic Standard Advanced Kits continued Signal Development with Chemiluminescent HRP Substrate continued Working Solution L00204C L00205C L00241 L00242 Preparation and L00228 and L00243 Reagent A 1 5 ml 1 0 ml Reagent B 1 5 ml 2 0 ml Total Volume 3 0 ml 3 0 ml Drain the excess wash solution from the membrane by holding the membrane vertically with forceps and touching the edge against a tissue Place the membrane on a clean flat surface and cover the membrane with working solution Incubate for three minutes at room temperature Place the membrane on a soft clean tissue Use another tissue to remove excess working solution Wrap the membrane in a clean piece of plastic film Expose to a sheet of film not provided for 30 seconds and then develop Repeat with different exposure time to find the best results An imager capable of detecting chemiluminescent signals can also be used to record the results Signal Development with TMB Substrate ChromoSensor One Solution TMB Substrate is a ready to use working solution and 0 05 ml is sufficient to cover 1 cm of membrane Drain the excess wash solution from the memb
17. han 2 yg of antibody per lane Theoretically 2 ug of antibody can pull down 1 33 ug of a 50 kDa antigen 3 If using a mouse L00232 or goat kit L00233 with Protein A G or A G MagBeads use the Protein A amp G blocker to prevent leaked protein A G or A G from interfering with the Western results If using a rabbit kit L00231 use Protein G blocker to prevent leaked protein G or A G from interfering with the western results Protein A does not affect the Western results in the case of rabbit antibodies All the kits are optimized to block up to 50 ng of protein A G or A G per lane Reagents not provided Primary antibodies Affinity purified antibodies are recommended Rabbit polyclonal antibodies should be whole molecule Fab fraction gives a significantly low signal GenScript has a complete portfolio of antibodies for signal pathways and other applications It may be viewed online here http Awww genscript com cgi bin products rec_antibody cgi Before use prepare the following 1X wash solution Dilute 25 ml of 5X wash solution with 100 ml of distilled or filtered water to make 125 ml of 1X wash solution If any precipitate forms in the 5X wash solution during storage incubate the bottle in a warm or hot water bath up to 50 C with occasional mixing until all the precipitate disappears Use 15 ml of 1X wash solution for each rinse and 20 ml of 1X wash solution for each wash 10 ONE HOUR Western Detection System
18. he corresponding fluorescent dye labeled secondary antibody the amount of secondary antibody is 50 of the primary antibody used to the same tube Vortex Mixture 1 gently for a few seconds and centrifuge briefly Incubate all the Mixture 1 s in the dark at room temperature for at least 40 minutes Refer to manufacturer s recommendations of appropriate amounts of antibody 18 ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western Multiplex Fluorescent Kit continued Pretreat Membrane Just before the protein transfer from gel to membrane is complete mix 10 ml of Pretreat Solution A with 10 ml of Pretreat Solution B in a plastic container Western Blot Box GenScript M00103 to make the pretreat solution mixture Always prepare and use a fresh solution mixture Place the membrane directly in the pretreat solution mixture and incubate on a shaker for five minutes at room temperature After incubation rinse the membrane twice with 15 ml of 1X wash solution Final Incubation of Pretreated Membrane a Just after setting up the pre treatment step add 1 ml of WB M to each of the Mixture 1 s and mix well by inverting the tubes several times Incubate all the tubes at room temperature for 5 minutes Then add all of the Mixture 1 s one by one to appropriate volume of WB M in a Western blot box GenScript Western Blot Box Black M00100 or M00103 and mix well The to
19. ng For mouse and goat kits If Protein A G or A G MagBeads is used during immunoprecipitation dilute 100 ul of Protein A amp G blocker with 10 ml of 1X wash solution and incubate the membrane from step 2 in this diluted blocker on a shaker for five minutes at room temperature Do not wash or rinse For rabbit kits If Protein G or A G MagBeads is used during immunoprecipitation first add Mixture 2 to 10 ml of IP WB 3 and then add 100 ul of Protein G blocker directly to the combined solution Mix well 11 ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR IP Western Kits continued Final Incubation of Pretreated Membrane a Add Mixture 2 to 10 ml of IP WB 3 in a plastic container and mix well Incubate the membrane in the IP WB 3 containing Mixture 2 ona shaker for 40 minutes at room temperature Rinse the membrane once with 15 ml of 1X wash solution Wash the membrane three times on a shaker for five minutes each with 20 ml of 1X wash solution Use a clean container for each rinse and wash step to avoid carryover contamination and to reduce background Signal Development Mix 1 5 ml of Reagent A with 1 5 ml of Reagent B by vortexing for a few seconds to make the working solution Use 0 1 ml of the working solution per cm of membrane The working solution is stable for several hours at room temperature when protected from light Drain the excess wash solution f
20. ng the primary antibody with WB 1 in a microcentrifuge tube Vortex Mixture 1 gently for a few seconds and centrifuge briefly Incubate Mixture 1 at room temperature for at least 40 minutes Mixture 1 L00204 L00205 L00204T L00205T L00204C L00205C L00241 L00242 Preparation and L00399 and L00228T and L00228 and L00243 WB 1 Solution 20 100 ul 50 100 ul 20 100 ul 5 25 ul Primary Antibody 2 10 ug 5 10 ug 2 10 ug 0 5 2 5 ug Ratio of WB 1 10 ul 1 yg 10 ul 1 ug 10 ul 1 ug 10 ul 1 ug Antibody For Antibody without Mix 5 ug of Ab Mix 5 ug of Ab Mix 5 ug of Ab Mix 1 ug of Ab Known Titer with 50 pl WB 1 with 50 pl WB 1 with 50 ul WB 1 with 10 ul WB 1 Refer to manufacturer s recommendations of appropriate amounts of antibody With ONE HOUR Western Advanced Kits use 1 4 to 1 2 of the recommended amount For antibodies without known titers start with 1 ug for Advanced Western Kits and 5 yg for other ONE HOUR Western Kits ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western Basic Standard Advanced Kits continued Pretreat Membrane Just before the protein transfer from gel to membrane is complete mix 10 ml of Pretreat Solution A with 10 ml of Pretreat Solution B in a plastic container Western wash box GenScript M00100 to make the pretreat solution mixture Always prepare and use fresh solution mixture Place the membrane directly in the pretrea
21. rane by holding the membrane vertically with forceps and touching the edge against a tissue Place the membrane on a clean plate and cover it with TMB Incubate for 5 to 10 minutes at room temperature until the desired color intensity is reached Stop the reaction by rinsing the membrane three times for thirty seconds each in 20 ml of deionized water Drain off the excess water and transfer the membrane to a piece of paper towel Air dry the membrane in a dark place ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western Basic Standard Advanced Kits continued Examples Comparison of the two ONE HOUR Western Kits of different sensitivities using monoclonal antibodies Two similar blots were processed with the same procedures using different ONE HOUR Western Kits Standard L00205C and Advanced L00242 10 ug and 2 5 ug of THE Anti GST Monoclonal Antibody Mouse GenScript A00865 respectively were used with these two kits to detect GST protein The results are shown in Figure 1 Standard Kit 3 60 1 20 0 41 0 13 0 045 Advanced Kit 1 2 3 4 5 6 7 tie e lt GST Loading 33 0 11 0 3 60 1 20 0 41 0 13 0 045 GST ng Figure 1 Western blots for the detection of GST protein using different ONE HOUR Western Kits Standard LO0205C and Advanced L00242 33 0 11 0 3 60 1 20 0 41 0 13 and 0 045 ng of GST protein were loaded onto Lane 1 2 3 4 5
22. rom the membrane by holding the membrane vertically with forceps and touching the edge against a tissue Place the membrane on a clean flat surface and cover the membrane with working solution Incubate for three minutes at room temperature Place the membrane on a soft clean tissue Use another tissue to remove excess working solution Wrap the membrane in a clean piece of plastic film Expose to a sheet of film for one minute and then develop Repeat with different exposure times for best results An imager capable of detecting chemiluminescent signals can also be used to record the results 12 ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR IP Western Kits continued Examples ProteinA Rabbit Ab Protein G Comparison of ONE HOUR IP Western blot and classical Western blot using rabbit primary antibody Western IP Western 123 4 123 4 Protein A 50 ng 50 ng Protein G 50 ng 50 ng Rabbit Ab 2ug 2ug 20ng 8ng Figure 1 Western blot detection of GST protein by both classical western and ONE HOUR IP Western using kit L00231 Both blots are developed using Rabbit Anti GST tag Polyclonal Antibody GenScript A00097 and the LumiSensor Chemiluminescent HRP Substrate included in kit L00231 Comparison of ONE HOUR IP Western blot and classical western blot using mouse primary antibody Western IP Western 123 4 123 4 ProteinA Ab H
23. se and wash step Wear gloves and use clean forceps to handle membranes There is cross reaction between primary antibody and secondary antibody The WB M solution containing all the Mixture 1 s is not mixed well Add Mixture 1 with 1 ml of WB M added one by one to WB M solution Mix well after each addition 294 ONE HOUR Western Detection System www genscript com Technical Support Web Resources Visit the GenScript Web site at www genscript com for 1 Technical resoures including manuals MSDS FAQ etc 2 Online 2010 2011 Product Catalog 3 Additional promotions and special offers Contact Us GenScript USA Inc 860 Centennial Ave Piscataway NJ 08854 Tel 732 885 9188 732 885 9688 Fax 732 210 0262 732 885 5878 Email product genscript com 25 ONE HOUR Western Detection System www genscript com Patent Pending Limited Use Label license This product may be the subject of one or more patents filed by GenScript USA Inc The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials ma
24. se a clean container for each wash to reduce background Imaging or Scanning After final wash transfer the membrane to a container containing 20 ml of distilled or filtered water Rinse the membrane for 1 minute and then scan the membrane on a LI COR Odyssey Infrared Imaging Systems following the Odyssey Operation Manual 16 ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western Fluorescent Kit continued Examples 1 Fluorescent Western blot detection of GST tag Antibody pAb Rabbit GenScript A00097 1 2 3 4 5 6 7 8 GST Protein ng 50 0 25 0 12 5 6 25 3 12 1 56 0 78 0 39 Figure 1 Fluorescent Western blots for the detection of GST protein using the ONE HOUR Western Fluorescent Kit L00397 50 0 25 0 12 5 6 25 3 12 1 56 0 78 and 0 39 ng of GST protein were loaded into Lane 1 2 3 4 5 6 7 and 8 respectively 2 Fluorescent Western blot detection of GAPDH Antibody pAb Goat GenScript A00191 1 2 3 4 5 6 7 8 HeLa cell lysate ug 5 0 2 5 1 25 0 62 0 31 0 16 0 08 0 04 Figure 2 Fluorescent Western blots for the detection of GAPDH using the ONE HOUR Western Fluorescent Kit L00397 5 0 2 5 1 25 0 62 0 31 0 16 0 08 and 0 04 ug of Hela cell lysate were loaded into Lane 1 2 3 4 5 6 7 and 8 respectively sita ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western
25. t solution mixture and incubate on a shaker for five minutes at room temperature After incubation rinse the membrane twice with 15 ml of 1X wash solution Final Incubation of Pretreated Membrane a Add Mixture 1 to 10 ml of WB 2 in a Western blot box and mix well Incubate the membrane in this solution WB 2 containing Mixture 1 on a shaker at room temperature for 40 minutes This solution WB 2 containing Mixture 1 may be recovered and reused up to three times if stored at 4 C However this may cause variations to arise due to changes in antibody concentration and carryover contamination b Rinse the membrane once with 15 ml of 1X wash solution Wash the membrane on a shaker three times for ten minutes each with 20 ml of 1X wash solution When using the TMB substrate wash the membrane three times for just five minutes each with 20 ml of 1X wash solution Use a clean container for each wash step to avoid carryover contamination and to reduce background Signal Development with Chemiluminescent HRP Substrate a When using LumiSensor Chemiluminescent HRP Substrate mix 1 5 ml of Reagent A with 1 5 ml of Reagent B by vortexing for a few seconds to make the working solution When using LumiSensor Super Chemiluminescent HRP Substrate mix 1 0 ml of reagent A with 2 0 ml of reagent B by vortexing for a few seconds to make the working solution Anout 0 05 ml of the working solution is sufficient to cover 1 cm of membrane Wh
26. tainer for each rinse and wash step Wear gloves and use clean forceps to handle membranes 393 ONE HOUR Western Detection System www genscript com Troubleshooting continued ONE HOUR Multiplex Fluorescent Kit Problem Probable Cause Solution The signal is Too little protein is loaded Load more protein s onto the weak or invisible SDS PAGE gel There is poor transfer efficiency Optimize the transfer time and or the electrical current Make sure that there are no air bubbles between the membrane and the gel The primary antibody has a low affinity for the antigen Increase the incubation time of the membrane in WB 2 containing Mixture 1 Increasing antibody concentration can also improve signal The primary antibody has a low affinity for the antigen Reducing wash time can increase the signal for low affinity antibody Instead of washing for 10 min x 3 wash for 5 min x 3 to increase signal There is high background Too much primary antibody was used Reduce the amount of primary antibody and reduce WB 1 accordingly The primary antibody has non specific binding or cross reactivity with the blocking reagent Use an alternate Pretreat A b M01057 The wash time is too short Adding additional washing steps can further decrease background The equipment or reagents have become contaminated Use a clean container for each rin
27. tal volume of the WB M solution should be 10 ml For example if 2 ml of WB M are already used to make 2 Mixture 1 s another 8 ml is needed to make the final solution Incubate the membrane in this solution WB M containing all the Mixture 1 s on a shaker at RT for 40 minutes Protect box or bag from light during incubation b Rinse the membrane once with 15 ml of 1X wash solution Wash the membrane on a shaker three times for ten minutes each with 20 ml of 1X wash solution Protect box or bag from light during wash Use a clean container for each wash to reduce background Imaging or Scanning After final wash transfer the membrane to a container containing 20 ml of distilled or filtered water Rinse the membrane for 1 minute and then scan the membrane on a LI COR Odyssey Infrared Imaging Systems following the Odyssey Operation Manual 19 ONE HOUR Western Detection System www genscript com Protocols continued ONE HOUR Western Multiplex Fluorescent Kit continued Examples 1 Multiplex Fluorescent Western blot detection of four proteins on the same membrane Hela cell lysate was spiked with GST protein as shown in Figure 1 All the primary antibodies and secondary antibodies are listed in the following table Antigens Primary Antibodies Amount Secondary Antibodies Amount a Tubulin Mouse Anti a Tubulin 6 ug IRDye 680 Donkey Anti Mouse 3 ug Monoclonal Antibody LI COR 926 3222
28. tandard Kit Rabbit L00204C ONE HOUR Western Standard Kit Mouse L00205C ONE HOUR Western Standard Kit Goat L00228 ONE HOUR Western Standard Kit with TMB Rabbit L00204T ONE HOUR Western Standard Kit with TMB Mouse L00205T ONE HOUR Western Standard Kit with TMB Goat L00228T ONE HOUR Western Advanced Kit Rabbit L00241 ONE HOUR Western Advanced Kit Mouse L00242 ONE HOUR Western Advanced Kit Goat L00243 ONE HOUR IP Western Kit Rabbit L00231 ONE HOUR IP Western Kit Mouse L00232 ONE HOUR IP Western Kit Goat L00233 ONE HOUR Western Fluorescent Kit L00397 ONE HOUR Western Multiplex Fluorescent Kit L00398 Contents ONE HOUR Western Detection Kits Components Basic Kits Standard Kits Advanced Kits With TMB Standard Pretreat Solution A 50 ml 50ml 50 ml 50 ml Pretreat Solution B 50 ml 50 ml 50 ml 50 ml WB 1 Solution 0 5 ml 0 5 ml 0 5 ml 0 5 ml WB 2 Solution 50 ml 50 ml 50 ml 50 ml 5X Wash Solution 125 ml 125 ml 125 ml 125 ml WestClear Nitrocellulose Membrane 5 sheets 5 sheets 5 sheets 0 2 um 7 5 x 8 cm ChromoSensor One 15 ml Solution TMB Substrate LumiSensor Chemiluminescent 2x7 5 ml HRP Substrate LumiSensor Super Chemiluminescent 5 10ml HRP Substrate User Manual 1 1 1 1 ONE HOUR Western Detection System www genscript com Kit Contents continued ONE HOUR Western Component Fluorescent Kit Multiplex Fluorescent

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