Lightgen HL Chemiluminescent Substrate User`s Manual
Contents
1. Iml Total 2ml m y lagene Lightgen HL User s Manual 3 Place the membrane on a leveled and sturdy hydrophobic surface such as on a piece of a parafilm with a piece of glass under Cover the entire membrane evenly with the substrate mixture 4 Make sure the membrane is covered with the substrate mixture evenly and then put the membrane with substrate solution on it into the imager 5 Follow the detailed instructions in the Cool Imager user s manual In general 2 5 minutes of capturing should be sufficient to obtain a chemiluminescent image 6 Adjust the capturing time to obtain optimal images 7 Use the Cool Imager Software to analyze and quantify the image 8 Refer to the user s manual of Cool Imager for all the details and other functions 2 Detection by exposing to X rays films When using X ray films make sure a dark room and a X ray film developer are available have enough X ray films chemicals and reagents 1 Turn on and pre warm the X ray film developer 2 Mix enough amount of substrate mixture refer to diction D 1 2 For example each 9 X 7 cm membrane needs at least 2 0 ml total volume of the mixture 3 Cover the entire membrane evenly with the substrate mixture and let it react at room temperature for 1 2 minutes 4 After the reaction discard the substrate on the membrane and then cover it with a clear plastic film 5 Inthe dark room put a sheet of X ray film on the wrapped2. Anal Biochem 240 306 308 4 Walker G R et al 1995 SuperSignal CL HRP A new enhanced chemiluminescent substrate for the development of the horseradish peroxide label in Western blotting applications J of NIH Research 7 76 ECL a trademark of Amersham Pharmacia Biotech LumiGlo is registered trademark of KPL Inc Western Lightning is a trademark of PerkinElmer Life Sciences Renaissance is a trademark of NEN Life Science Products Tween is a registered trademark of ICI Americas Cool Imager and Lightgen HL are trademarks of Viagene Biotech Inc Please visit us at www viagene com for latest news and products information
3. NA and Insufficient hybridization probes Inefficient protein transfer Optimize protein transfer Viagene Lightgen HL User s Manual Low HRP or substrate activity Increase HRP conjugates High background Too much HRP Dilute HRP conjugate more Insufficient blocking Optimize blocking conditions Inappropriate blocking buffer Try another blocking buffer Inadequate washing Increase washing time and or volume of washing solution Film or CCD camera have been overexposed Decrease exposure time Concentration of antigen or antibody is too high Reduce amount of antigen or antibody used in experiments White spots on the Inefficient protein transfer Optimize protein transfer procedure membrane randomly Unevenly hydrated membrane Follow manufacturer s recommendations Bubbles between film and the membrane Remove all bubbles before film exposure Speckles on the film background Aggregate formation in the HRP conjugate Filter conjugate through a 0 2 um filter Nonspecific bands Too much HRP in the system Dilute HRP conjugate at least 10 fold SDS caused nonspecific binding to protein bands Do not use SDS during immunoassay procedure G Related Viagene Products IMGR002 Cool Imager chemiluminescent imager workstation TFDET001 Non radioactive NF KB EMSA Kit 50 reactions TFDET002 No
4. Viagene Lightgen HL User s Manual Lightgen HL Chemiluminescent Substrate User s Manual This instruction is suitable for following products Catalog No Name Description Volume High Sensitive CHHL 100 Lightgen HL100 mae 100 ml Chemiluminescent Substrate High Sensitive CHHL 050 Lightgen HL50 maa 50 ml Chemiluminescent Substrate es High Sensiti CHHL 020 Lightgen HL20 8 gt ensiive 20 ml Chemiluminescent Substrate Viagene Biotech Inc 3802 Spectrum Blvd Suite 128B Tampa Florida 33612 Tel 813 902 2209 Fax 813 903 0649 E mail viagene hotmail com Viagene Lightgen HL User s Manual TABLE OF CONTENTS A Introduction apo uscesconeacasencmcssedtsdincnenunsttcesseiie candies 3 B Kitcomponents scanietat neocon wiaemeentomanme 3 c Additional material required ou eeeeeeeeeeteeees 4 Da Procedures sraicecrastonatienlnseaee 4 E Preca tio ns saccericacvaiseevisneviciwrmesssedepeticcdsawess 5 F Troubleshooting eessesseessesesesessseesssessrssseresssessssssee 6 G Related Viagene Products eececeesseeeeeere eee 7 H Waranty sorsero ina ea a 8 T ARCTIC ECC SS snared sGorresattessoaiececutensceaeadenariienseneenndentutnnneces 8 2007 Viagene Biotech Inc All rights reserved m y lagene Lightgen HL User s Manual A Introduction Lightgen HL is a chemiluminescent substrate with extremely high sensitivit
5. l C Additional materials required not included Lightgen HL is designed for detecting the activities of HRP on nitrocellulose PVDF or Nylon membranes Before proceeding please have the following materials ready 1 Any one of the following completely reacted membranes In Western Blots membranes should have been incubated with primary and HRP conjugated secondary antibodies They should be ready for chemiluminescent detecting In Northern and Southern Hybridization membrane should have been hybridized with HRP conjugated probes and ready for chemiluminescent detecting In EMSA membrane should been incubated with detecting reagents and ready for chemiluminescent detecting 2 A CCD image detector specifically for pickup Chemiluminescent signals such as Viagene s Cool Imager or X rays films a film developer and related chemicals and reagents D Procedures 1 Detection with a CCD Imager A CCD imager should have high sensitivity and capable of capturing all the needed data in 5 10 minutes Viagene s Cool Imager is an example of choice to capture images for Western Blots Northern and Southern hybridizations or EMSA Detecting and analyzing images with the CCD imager is much easier than X Ray films Following operation is for Viagene s Cool Imager 1 Turn on Cool Imager and pre cool the system for 1 2 minutes 2 Make application mixture for each 7 X 9 cm membrane Solution A Iml Solution B
6. membrane Since it is difficult to pre determine the exposure time for each experiment it may be necessary to try different exposure time to obtain best results E Precautions 1 In order to obtain best results it is important to use high quality experiment materials equipments such as samples quality probes and antibodies concentration membrane quality etc 2 Because Lightgen HL has extremely high sensitivity it will be necessary too optimize the dilution of antibodies hybridization probes or HRP conjugates In the experiments using Lightgen HL only 1 3 to 1 5 of amounts of samples antibodies or probes used in the experiment with the substrates rather than Lightgen HL is required 3 Before the experiment it is recommended to check the concentration of probes and antibodies by performing the dot blot analysis 4 All the containers used in chemiluminescent experiments should be cleaned thoroughly Always wear gloves or use cleaned forceps to handle membranes to avoid high background Don t let membranes m y lagene Lightgen HL User s Manual dry out during experiments Choose an appropriate blocking buffer There is not a certain generic buffer for all Western blot Northern and Southern hybridization and EMSA Choosing the appropriate blocking buffer may increase chemiluminescent sensitivity and reduces nonspecific background Avoid using milk in blocking buffer when using avidin biotin systems becau
7. n radioactive STAT EMSA Kit 50 reactions TFDET003 Non radioactive STAT3 EMSA Kit 50 reactions TFDET004 Non radioactive STATS EMSA Kit 50 reactions TFDET005 Non radioactive AP 1 EMSA Kit 50 reactions TFDET006 Non radioactive HIF 1 EMSA Kit 50 reactions Viagene Lightgen HL User s Manual H Warranty Thank you for choosing Viagene products Viagene Biotech Inc guarantees that Lightgen will perform as they are intended for and indicated in each manual Products are warranted for 1 year from the date of purchase unless otherwise specified During the warranty period if problems arise Viagene will be responsible for products exchange or return All Viagene products are for research purposes only No resale distribution or sale modified products of Viagene Biotech unless written permission is granted Viagene Biotech Inc strives for complete customer satisfaction if you are not satisfied with Viagene s products would like to give us comments please contact Viagene Biotech customer service I Warranty 1 CRC Handbook of Immunoblotting of Proteins Volume 1 Technical Description Eds Ole J Bjerrum Ph D M D and Niels H H Heegaard M D CRC Press Inc Boca Raton FL 1988 2 Kaufmann S H et al 1987 The erasable Western blot Anal Biochem 161 89 95 3 Mattson D L and Bellehumeur T G 1996 Comparison of three dhemiluminescent horseradish peroxidase substrates for immunoblotting
8. se milk contains variable amounts of endogenous biotin Adding Tween 20 or other high quality detergent to the blocking and washing buffers may reduce nonspecific background Washing and blocking buffers cannot contain sodium azide as it will inhibit HRP activity Because the chemiluminescence decays over time the first 5 30 minute reaction of HRP and chemiluminescent substrate produces the strongest chemiluminescence In order to obtain best results capturing chemiluminescent images with different exposure time is necessary During detection of chemiluminescent images any movement of CCD camera or X ray films on the membrane should be avoided to obtain best results Solution A and B should be stored in their own bottles at 4 C for long term storage At room temperature the substrate mixture is stable for 72 hours and exposure it to direct light may reduce its stability However short time exposure to lab lighting has no effects on substrate solutions F Troubleshooting Problems Possible Causes Recommendation White spots in the center of dark chemiluminescent bands Too much HRP on sample area digests the substrate quickly and Dilute Probes antibodies or diminish chemiluminescence HRP conjugates more Brown or yellow spots on the membrane Very weak or no signal Insufficient interaction of antigen Increase amount of antigen or amp antibody antibody Increase amount of RNA D
9. y used for detecting Horseradish Peroxidase in Northern Southern Hybridization Electrophoretic Mobility Shift Assay EMSA and Western blotting HRP by a digital imager When incubating with HRP it emits a strong blue light which can be detected by a CCD imager or X Ray films Detecting sensitivity can reach to femtogram level of HRP when Lightgen HL is used Lightgen HL is very easy to use highly sensitive and stable suitable for experiments which use nitrocellulose PVDF or Nylon membranes Since Lightgen HL is much more sensitive than other chemiluminescent substrates in the market such as ECL LumiGLO Renaissance Western Lightnight it will be necessary to optimize the dilution of antibodies hybridization probes or HRP conjugates to obtain best results Please refer to the procedure section for details B Kit components 1 100 ml Substrate Kit CHHL100 Lightgen HL Solution A 50 ml Lightgen HL Solution B 50 ml Sufficient for 60 300 pieces of 7 x 8 cm membrane or 3 000 15 000 cm membrane 2 50 ml Substrate Kit CHHL050 Lightgen HL Solution A 25 ml Lightgen HL Solution B 25 ml Sufficient for 30 150 pieces of 7 x 8 cm membrane or 1 500 7 500 cm membrane 3 20 ml Substrate Kit CHHL020 Lightgen HL Solution A 10 ml Lightgen HL Solution B 10 ml Sufficient for 12 60 pieces of 7 x 8 cm membrane or 600 300 cm membrane Viagene Lightgen HL User s Manua
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