Lightgen CL Chemiluminescent Substrate User`s Manual
Contents
1. Viagene Lightgen CL User s Manual Lightgen CL Chemiluminescent Substrate User s Manual This instruction is suitable for following products Catalog No Name Description Volume CHCL 250 Lightgen CL250 High Sensitive 250 ml m TEM Chemiluminescent Substrate CHCL 125 Lightgen CLi2s_ Hist Sensitive 125 ml m eee Chemiluminescent Substrate CHCL 050 Lightgen CL050 High Sensitive m Im Chemiluminescent Substrate Viagene Biotech Inc 3802 Spectrum Blvd Suite 126 Tampa FL 33612 Tel 813 902 2209 m Fax 813 903 0649 E mail info viagene com Viagene Lightgen CL User s Manual TABLE OF CONTENTS A Introducti sseos enina eare AREE 3 B Kitcomponents sesseeeeessseeesseesssssreesssrsssressssresssee 3 C Additional material required usus 3 D PEODDQU ES Susssesb S MRNA MUN RR RU EE 4 E Preca tions quoesussespodt diae IONS UE as 5 E Troubleshooting asadesceespesa Repente ER SHEER VR PEE ex tants 6 G Related Viagene Products sess 7 MEME Waranty ATRIDES e TES 7 L References Acc 7 20070 Viagene Biotech Inc All rights reserved Viagene Lightgen CL User s Manual A Introduction Lightgen CL is a chemiluminescent substrate with high sensitivity using for detecting Horseradish Peroxidase in Western blot detection and Electrophoretic Mobility Shift Assay EMSA When incubating with HRP it em2. a CCD Imager For capturing chemiluminescent images a CCD imager must have very high sensitivity and capacity to handle thousands of images in a short time 1 15 minutes Viagene s Cool Imager is a good example of choice for capturing images of Western Blots Northern Southern hybridizations and EMSA Detecting and analyzing images with the CCD imager is much easier than X Ray films Following operation manual is for Viagene s Cool Imager 1 2 3 4 Turn on Cool Imager and pre cool the system for 1 2 minutes 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 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 Make sure the membrane is covered with the substrate mixture evenly and then put the membrane with substrate solution into the imager Viagene Lightgen CL User s Manual 5 Follow the detail instructions in Cool Imager user s manual In general 4 10 minutes of capturing should be sufficient to obtain a chemiluminescent image Adjust the capturing time to obtain optimal images 6 Use the Cool Imager Software to analyze and quantify the image 7 Refer to the user s manual of Cool Imager for all the details and other functions E Precautions In order t
3. ane 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 Viagene Lightgen CL User s Manual 2 X rays films film developer and related chemicals and reagents or a CCD image detector specifically for pickup Chemiluminescent signals such as Viagene s Cool Imager D Procedures l Detection by exposing to X rays films When using X rays films make sure a dark room and a X ray film developer are available also have enough X ray films chemicals and reagents 1 2 3 4 5 Turn on and pre warm the X ray film developer Make application mixture for membranes Solution A 1ml Solution B 1 ml Total 2 ml 2 ml of substrate mixture is enough to cover a mini gel membrane A large membrane needs more substrate mixture Averagely 0 03ml of substrate mixture is enough to cover 1 cm of NC PVDF or Nylon membranes Cover the entire membrane evenly with the substrate mixture and let it reacts at room temperature for 1 2 minutes After the reaction discard the substrate on the membrane and then cover it with a clear plastic film In the dark room put a sheet of X rays film on the wrapped 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 2 Detection with
4. d 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 or would like to give us comments please contact Viagene Biotech customer service I References 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 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 Viagene Lightgen CL User s Manual 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 CL are t
5. its a blue light which can be detected by X Ray films or a CCD imager Detecting sensitivity can reach to nanogram level of HRP on nitrocellulose NC PVDF or Nylon membranes It may 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 Lightgen CL is easy to use by combine equal amounts of Solution A and B B Kit components Lightgen CL 250 ml Substrate Kit CHCL 250 Lightgen CL Solution A 125 ml Lightgen CL Solution B 125 ml Sufficient for 150 pieces of 7 x 8 cm membrane or 8300 cm membrane 125 ml Substrate Kit CHCL 100 Lightgen CL Solution A 62 5 ml Lightgen CL Solution B 62 5 ml Sufficient for 60 pieces of 7 x 8 cm membrane or 4000 cm membrane 50 ml Substrate Kit CHCL 050 Lightgen CL Solution A 25 ml Lightgen CL Solution B 25 ml Sufficient for 30 pieces of 7 x 8 cm membrane or 1600 cm membrane C Additional materials required not included Lightgen CL is designed for detecting the activities of HRP on nitrocellulose PVDF or Nylon membranes Before proceeding please have the following materials ready l 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 membr
6. movement of CCD camera or X rays 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 Viagene Lightgen CL User s Manual Problems White spots in the center of dark chemiluminescent bands Brown or yellow spots on the membrane Possible Causes Too much HRP on sample area digests the substrate quickly and diminish chemiluminescence Recommendation Dilute Probes antibodies or HRP conjugates more Very weak or no signal Insufficient interaction of antigen amp antibody Increase amount of antigen or antibody Insufficient hybridization Increase amount of RNA DNA and probes Inefficient protein transfer Optimize protein transfer Low HRP or substrate activity Increase HRP conjugates High background Too much HRP Dilute HRP conjugate more Insufficient blocking Inappropriate blocking buffer Inadequate washing Optimize blocking conditions Try another blocking buffer Increase washing time and or volume of washing solution Film or CCD camera have been overexposed Concentration of antigen or antibody is too high Decrease e
7. o obtain best results it is important to use high quality experiment materials equipments such as samples quality probes and antibodies concentration membrane quality etc Before the experiment it is recommended to check the concentration of probes and antibodies by performing the dot blot analysis 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 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 the avidin biotin systems because 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 imaging any
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9. xposure time Reduce amount of antigen or antibody used in experiments White spots on the membrane randomly Inefficient protein transfer Optimize protein transfer procedure Unevenly hydrated membrane Follow manufacturer s recommendations Bubbles between film and the membrane Remove all bubbles before film exposure Viagene Lightgen CL User s Manual Speckles on the film background Aggregate formation in the HRP conjugate Filter conjugates 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 TFDETO001 Non radioactive NF KB EMSA Kit 40 reactions TFDET002 Non radioactive STAT EMSA Kit 40 reactions TFDET003 Non radioactive STAT3 EMSA Kit 40 reactions TFDET004 Non radioactive STATS EMSA Kit 40 reactions TFDETO005 Non radioactive AP 1 EMSA Kit 40 reactions TFDETO006 Non radioactive HIF 1 EMSA Kit 40 reactions H Warranty Thank you for choosing Viagene products Viagene Biotech Inc guarantees that EMSA kit 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 perio
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