Cell homogenates were studied by Standard Western blot techniques. Briefly, 25 μg of homogenate was separated on a 10% acrylamide gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ) with a semidry transfer apparatus. The membranes were blocked with Tris Buffered Saline-5% milk and then probed with anti caspase-3, anti TNF-α, anti-IFN-γ and anti- IL-2 (sc-2710281, sc-1350, sc-59993 and sc-48545; 1:200, 1:100, 1:100 and 1:100, respectively, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibody was detected with horseradish peroxidase-conjugated anti-mouse secondary antibody (sc-2005, diluted 1:5000, 1:2000, 1:1000 and 1:2000 for caspase-3, TNF-α, IFN-γ and IL-2, respectively, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were washed, and blots were developed using an enhanced chemiluminescence Western blotting detection kit(ECL Plus kit, Amersham Biosciences RPN2132, USA) and exposed to X-ray film (Sigma C4729-1EA, Sigma Z363006-50) for 2–30 s. Molecular weight standards (See Blue® Plus2, LC 5925, Life Technologies, USA) were used to determine molecular weights of the visualized bands.
Sc 59993
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-59993 is a laboratory product offered by Santa Cruz Biotechnology. It is a research-use-only tool intended for scientific investigation. The core function of this product is to facilitate the study and analysis of biological samples and processes. No further details on the intended use or application of Sc-59993 can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus kit, by Cytiva (3 mentions)
Sc 1350, by Santa Cruz Biotechnology (3 mentions)
Hybond p, by Cytiva (3 mentions)
Seeblue plus2, by Thermo Fisher Scientific (2 mentions)
Sc 2710281, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody sc 2005, by Santa Cruz Biotechnology (2 mentions)
Protein assay, by Bio-Rad (1 mentions)
Sc 2035, by Santa Cruz Biotechnology (1 mentions)
3 protocols using sc 59993
1
Western Blot Analysis of Inflammatory Markers
2
Quantifying Apoptosis Signaling Proteins
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To find out whether changes in caspase-3 activity and concentrations of TNF-α and IFN-γ were due to changes in protein content, cell homogenates were studied by standard western blot techniques. Briefly, 25 μg of homogenate protein were separated on a 10% acrylamide gel by SDS-PAGE and then transferred to poly vinylidene difluoride membranes (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ, USA) with a semidry transfer apparatus. The membranes were blocked with TBS-5% milk and then probed with anti caspase-3, anti -TNF-α, and anti-IFN-γ (sc-2710281, sc-1350, and sc-59993; 1:200, 1:100 and 1:100, respectively, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibody was detected with horseradish peroxidase-conjugated antimouse secondary antibody (sc-2005, diluted 1:5000, 1:2000 and 1:1000 for caspase-3, TNF-α and IFN-γ, respectively, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were washed and blots were developed using an enhanced chemiluminescence western blotting detection kit (ECL Plus kit, Amersham Biosciences RPN2132, USA) and exposed to x-ray film (Sigma C4729-1EA, Sigma Z363006-50) for 2-30 s. Molecular weight standarts (See Blue® Plus2, LC 5925, Life Technologies, USA) were used to determine molecular weights of the visualized bands.
3
Quantifying Apoptosis Markers in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the effects of EB on caspase-3, TNF-α, and IFN-g protein levels, cell homogenates were analyzed by standard western blotting techniques (Papi et al., 2013) (link). The protein concentration of the cell homogenates was assayed using the Bio-Rad protein assay, and 25-µg samples of homogenate protein were separated on a 10% acrylamide gel by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ, USA) using a semidry transfer apparatus. The membranes were blocked and then probed with anticaspase-3, anti-TNF-α, and anti-IFN-g antibodies (sc-2710281, sc-1350, and sc-59993, respectively, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibodies were diluted 1:100 and the horseradish peroxidase-conjugated mouse anti-human IgG secondary antibody (sc-2005 Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted 1:2000. The membranes were washed, and blots were developed using ECL Plus kit (Amersham Biosciences, Piscataway, NJ, USA) and exposed to X-ray film for 2-30 s. Molecular weight standards (sc-2035, Santa Cruz Biotechnology) were used to determine the molecular weights of visualized bands. Beta actin was used as a protein loading control.
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