Assay Kit (CPRG)
Contents
1. g Genlantis A Division of Gene Therapy Systems Inc Enhanced 6 Galactosidase Assay Kit CPRG RELATED PRODUCTS GenePORTER Transfection Reagent 75 reactions 7201007 GenePORTER Transfection Reagent 150 reactions 7201015 GenePORTER Transfection Reagent 750 reactions 1201075 Stop Buffer 55 ml 4 C GenePORTER 2 Transfection Reagent 75 reactions T202007 10X CPRG Substrate Stock 5x1ml 20 C GenePORTER 2 Transfection Reagent 150 reactions T202015 Chlorophenol red B D galactopyranoside GenePORTER 2 Transfection Reagent 750 reactions gWiz B galactosidase Expression Vector 25 ug P010200 A10200K A10300K 6 galactosidase Assay Kit ONPG X Gal Staining Kit Shipping Condition INTRODUCTION LacZ is a commonly used reporter gene in transfection experiments because the gene product B galactosidase is very stable resistant to proteolytic degradation and easy to assay Levels of active B galactosidase expression can be quickly measured by its catalytic hydrolysis of Chlorophenol red B D galactopyranoside CPRG substrate to a dark red product The assay kit provides all the required reagents and offers a rapid simple and sensitive method to quantify the enzyme expression in transfected cells e g transfected with Genlantis gWiz B gal vector The high sensitivity improves the measurement of B gal activity when the reporter gene expression is low USAGE e Transfect cells with a plasmid exp2. of cell lysate from non transfected cells negative control to a tube to control endogenous galactosidase activity Add 50 ul of Standard Dilution Buffer to each tube Prepare a serial dilution of f galactosidase E coll standards with Standard Dilution Buffer separately Transfer 50 ul of each standard to a fresh tube containing 100 ul cell lysate from a mock transfection The highest recommended amount of beta galactosidase is 200 000 pg 100 milliunits Adjust the standard curve to suit the specific experimental conditions such as cell type transfection reagent or plasmid vector 2X serial dilution of standard curve consisting of 8 points is recommended A dilution protocol example is shown in the section of 96 well plate assay Add 300 ul of 1X CPRG Substrate Solution to each tube Incubate the tubes at room temperature till the red color develops from approximately 10 minutes to 4 hours depending on the cell type Add 500 ul of Stop Solution to stop the reaction Final volume is 950 ul Read the absorbance at 5 0 595 nm with a spectrophotometer Quantify B galactosidase expression based on a linear standard curve Felgner J H et al Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations J Biol Chem 269 2550 2561 1994 Genlantis Toll Free 888 428 0558 e 858 457 1919 Web http www genlantis com Page 2 of 2
3. of Lysis Buffer depends on the size of the culture dishes used for transfection i e cell pellet size we recommend using incre a Incubate the cell lysate 10 15 minutes at room temperature by gently swirling the dishes several times to ensure 3 Incubate the dish 10 15 minutes at room temperature by complete lysis Proceed to the colorimetric assay or freeze swirling it slowly several times to ensure complete lysis the plate at 70 C until ready The culture dishes can be observed under a microscope to NOTE See NOTE above confirm that the cells are lysed completely OPTIONAL See OPTIONAL above 4 Proceed to Section C Below Sraccad ta Section C Below VKM021607 Genlantis Page 1 of 2 10190 Telesis Court San Diego CA 92121 Toll Free 888 428 0558 e 858 457 1919 Web http www genlantis com Enhanced Galactosidase Assay Kit CPRG Cat A10100K C Colorimetric CPRG Assay NOTE Dilute 10X CPRG stock to 1X with Substrate Buffer just before performing the colorimetric assay Unused 1X CPRG may be stored at 20 C for future use We recommend using 1X CPRG solution only 2 times after a freeze thaw cycle CAUTION Wear Gloves for manipulating the CPRG since it will stain exposed skin 96 well Microtiter Plate Assay 1 Thaw the dish tube or plate of lysed cells at room temperature If the transfection is performed with a 96 well plate perform the assay directly on the plate flat bottom only 2 Add 50 ul of Standa
4. rd NOTES Adjust the standard curve to suit the specific experimental conditions such as cell type transfection reagent or plasmid vector The dilutions for the standard curve must be prepared fresh each the assay 4 Add 50 ul of each sample well NOTE It may be necessary to dilute the cell lysate in 1X Lysis Buffer when transfection efficiency is very high In contrast when transfection efficiency is low reduce the volume of lysis buffer used to harvest the cells see description above or use up to 150 ul of cell extract for the colorimetric assay If the transfection is performed in a 96 well plate perform the assay directly on the plate 5 Prepare a blank by adding 50 ul of lysis buffer to a well Add also 50 ul of cell lysate from non transfected cells negative control to a well to control endogenous galactosidase activity VKM021607 10190 Telesis Court San Diego CA 92121 5 Thaw the cell lysate and transfer 100 ul to a fresh tube or 50 ul to a 96 well plate If a 96 well plate is used follow the protocol described above NOTE It may be necessary to dilute the cell lysate in 1X Lysis Buffer when transfection efficiency is very high In contrast when transfection efficiency is low reduce the volume of lysis buffer used to harvest the cells see description above or use up to 150 ul of cell extract for the colorimetric assay Prepare a blank by adding 100 ul of lysis buffer to a tube Add also 100 ul
5. rd Dilution Buffer to the wells of a 96 well plate except control wells which are set aside for a standard curve see below 3 Prepare a serial dilution of B galactosidase E coll Standards using the Standard Dilution Buffer separately A 50 ul aliquot of each point on the standard curve is transferred to the control wells of the plate the highest recommended amount of f galactosidase is 100 milliunits 100 000 200 000 pg 2X serial dilution of Standard curve consisting of 8 points is recommended A dilution protocol example is shown in the following table Add 100 ul of 1X CPRG Substrate Solution to each well Incubate plate at room temp until dark red color develops 10 minutes to 4 hours depending on cell type Read the absorbance at 570 595 nm with a microtiter spectrophotometer Stop solution is not required for this format since all wells are read simultaneously without a time gap Be sure that there are no bubbles present in the wells while reading because they interfere with the absorbance reading remove bubbles with a fine gauge needle tips or very weak gas flow Quantify B galactosidase expression based on a linear standard curve Macro assay g Standard Dilution Buffer miliunits Volume 3 4 200 ul 200 ul of 25 mu B gal standard 200 ul 200 ul of 12 5 mu B gal standard 200 ul 200 ul of 6 25 mu B gal standard 1 562 200 ul 200 ul of 3 125 mu B gal standard 200 ul 200 ul of 1 562 mu B gal standa
6. ressing LacZ gene e Lyse cells using lysis buffer e Transfer the lysate to a fresh tube or a microtiter plate Dilute the lysate if needed e Prepare a B galactosidase standard curve with standard dilution buffer e Add the substrate and monitor the color development at 570 595 nm e Calculate the expression levels based on a standard curve EXAMPLE PROTOCOLS NOTE Dilute 5X Lysis buffer to 1X with distilled deionized water before use Unused 1X Lysis Buffer may be stored at 4 C for future use NOTE A quick freeze thaw cycle freeze 1 2 hours at 20 C or 70 C then thaw at room temperature of the dish can be performed to improve lysis Keep dish frozen at 70 C until ready to proceed to the colorimetric assay OPTIONAL Before proceeding to the colorimetric assay the plate or dish can be centrifuged for 2 3 minutes to pellet the insoluble material Use the supernatant for the assay A Protocol For Adherent Cells 1 Aspirate the growth medium from the culture dish 24 72 hours after transfection use non transfected cells for control Optionally wash cells 1 time with 1X PBS 2 Add 1X Lysis Buffer to the culture dish Use the following Protocol For Suspension Cells recommended volumes depending on your culture dish Aspirate the supernatant 24 72 hours post transfection after centrifuging cells at 250 x g for 5 minutes Optionally wash cells 1 time with 1X PBS Resuspend the cell pellet in 1x Lysis Buffer The amount
Download Pdf Manuals
Related Search