The cells were washed in PBS and harvested in 200 μl RLB Buffer (Promega, E397A). 20 μl of this solution was mixed with 100μl detection solution (Beetle Lysis Juice, PJK) and measured using the luminometer Lumat LB 9507 (Berthold). Light units obtained from 10 s were related to the protein content. The protein content was measured with Nanodrop biophotometer (Eppendorff).
Rlb buffer
Manufactured by Promega
Sourced in United States
The RLB Buffer is a reagent used in the lysis of mammalian cells for the extraction of RNA, DNA, and proteins. It is designed to effectively disrupt cell membranes and release cellular contents while preserving the integrity of the target biomolecules.
Automatically generated - may contain errors
4 protocols using rlb buffer
1
Luminescence-Based Protein Quantification
2
Luciferase Assay in C. jejuni
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Expression of the luciferase in C. jejuni 81116 and racRS mutant strain harboring the pMA5-ggtprom-luc plasmids was measured as previously described (Bouwman et al., 2013 (link)). Briefly, overnight cultures were diluted to an OD550nm of 0.05 and grown for 7.5 h in HI with 50 mM KNO3 in an oxygen limiting atmosphere at 37°C. One milliliter of each culture was pelleted (8000 ×g, 5 min, 4°C) and suspended in 100 μL RLB buffer (Promega) supplemented with 0.5% Triton-X100. Suspensions were stored at −80°C for at least 30 min to disrupt the bacteria. Bacterial lysate (20 μL) was mixed with 50 μL of luciferase reagent (Promega) and RLU’s were measured immediately on a luminometer (TD20/20, Turner Designs). The data shown represents at least three independent experiments.
3
Luciferase Activity Assay Protocol
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The cells were washed in PBS and harvested in 250 μl RLB Buffer (Promega, E397A). 20 μl of this solution was mixed with 100 μl detection solution (Beetle Lysis Juice, PJK) and measured using the luminometer Lumat LB 9507 (Berthold). The protein content was measured with Nanodrop biophotometer (Eppendorff). Relative light units (RLU) obtained from 10 s were related to the protein content (RLU/mg).
4
Assessing IFN-β Promoter Activity
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HEK293 cells were seeded into 24-well plates in an amount of 1 × 105 cells/well. The next day, cells were transfected with 0.5 µg of plasmid IFN-Beta_pGL3 (Addgene) encoding luciferase under the control of IFN-β promoter and 0.5 µg of either plasmids encoding HCV NS5B, or HIV-1 RT, or inactivated HIV-1 RT, or empty vector pVax1 using 1 µL Lipofectamine LTX and 0.5 µL Plus Reagent. Twenty hours post transfection, cells were harvested, lysed using RLB buffer (Promega, Madison, WI, USA), centrifuged, and supernatant was assessed for luciferase activity using the Luciferase Assay System (Promega) as recommended by the manufacturer. Luminescence was measured on a luminometer (Promega).
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