With the help of RIPA lysis buffer (Beyotime, Shanghai, China), protein lysates for tissues and cells were prepared along with 1% (vol/vol) phosphatase inhibitors and phenylmethanesulfonyl fluoride (PMSF). Concentrations were estimated by using the Bicinchoninic Acid (BCA) Protein detection Kit (Beyotime, Shanghai, China). Electrophoresis was performed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinylidene difluoride (PVDF) membrane. Blocking with 5% (w/v) skims milk for 2h at room temperature (RT), followed by rabbit anti-mouse β-actin (cat. 20536-1-AP,1:1000, Proteintech), cGAS (cat.26416-1-AP,1:1000, Proteintech), p-STING (cat.50907T (Ser366,); cat.62912S1(Ser365,),1:1000, Cell Signaling Technology), STING (cat. 19851-1-AP,1:1000, Proteintech), p-TBK1 (cat. 5483 T,1:1000, Cell Signaling Technology), TBK1 (cat.28397-1-AP 1:1000, Proteintech) overnight at 4 °C, then incubate the secondary antibody (BA1054, 1:5000, Boster) for 1 h at RT. Furthermore, the levels of the internal reference gene grayscale values were used to estimate the target genes' protein expression levels.
Rabbit anti mouse β actin
Manufactured by Proteintech
Sourced in United States
Rabbit anti-mouse β-actin is a primary antibody that specifically recognizes the β-actin protein in mouse samples. It is commonly used in western blotting, immunohistochemistry, and other applications to detect and quantify the expression of this ubiquitous cytoskeletal protein.
Automatically generated - may contain errors
Lab products found in correlation
P sting, by Cell Signaling Technology (1 mentions)
Sting, by Proteintech (1 mentions)
P tbk1, by Cell Signaling Technology (1 mentions)
Ba1054, by Boster Bio (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein detection kit, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
2 protocols using rabbit anti mouse β actin
1
Protein Quantification and Western Blot
2
Quantitative analysis of gene and protein expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total RNA was extracted using Trizol Reagent. 500 ng of total RNA was reverse-transcribed using the PrimeScript RT Master Mix to produce cDNA. SYBR Green qPCR SuperMix was used to perform qPCR. Real-time PCR was carried out in LightCycler 480 using the forward and reverse primers shown in Table S1. PCR programming was set as pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5S, and annealing at 60 ℃ for 30s in 45 cycles. Data were calculated by 2 -ΔΔCT method with β-actin as internal reference.
Western blotting Tissue samples were crushed and incubated in RIPA lysis buffer to produce lysate. Protein concentrations were determined using a BCA kit (Beyotime Institute of Biotechnology, Jiangsu, China). Total protein (20 ng per sample) was separated using SDS-PAGE on a 10% polyacrylamide gel and then electrophoretically transferred onto a polyvinylidene di uoride membrane. The membranes were cut and incubated with the following speci c primary antibodies, rabbit anti-mouse Occludin (1:1000, Abcam, USA) and rabbit anti-mouse β-actin (1:10000, Proteintech, USA). Bands were detected using an ECL chemiluminescence system after treated with secondary antibodies.
Western blotting Tissue samples were crushed and incubated in RIPA lysis buffer to produce lysate. Protein concentrations were determined using a BCA kit (Beyotime Institute of Biotechnology, Jiangsu, China). Total protein (20 ng per sample) was separated using SDS-PAGE on a 10% polyacrylamide gel and then electrophoretically transferred onto a polyvinylidene di uoride membrane. The membranes were cut and incubated with the following speci c primary antibodies, rabbit anti-mouse Occludin (1:1000, Abcam, USA) and rabbit anti-mouse β-actin (1:10000, Proteintech, USA). Bands were detected using an ECL chemiluminescence system after treated with secondary antibodies.
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