Total proteins were extracted using a total protein extraction kit (Vazyme Biotech, Nanjing, China), and concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Biotechnology, Shanghai, China). A total of 20 μg of protein was separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated in blocking buffer containing 5% nonfat dry milk in Tris-buffered saline and Tween 20 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.05% Tween) for 1 h at room temperature. After incubating the membrane with primary antibody (rabbit anti-c-Kit antibody diluted at 1 : 3000) overnight at 4°C and horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibody (1 : 4000, Abcam) for 2 h at room temperature, protein-antibody complexes were visualized with an Enhanced Chemiluminescence Western Blotting Detection Kit (Beyotime Biotechnology, Shanghai, China) and analysis system (Bio-Rad). β-Actin was detected by the same method as a loading control.
Horseradish peroxidase hrp conjugated goat anti rabbit antibody
Manufactured by Abcam
Sourced in United States
Horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibody is a secondary antibody that binds to primary rabbit antibodies. The HRP enzyme label can be used to detect and quantify the presence of the target antigen recognized by the primary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Total protein extraction kit, by Vazyme (1 mentions)
Enhanced chemiluminescence western blotting detection kit, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Rage antibody, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Merck Group (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Amersham ecl plus kit, by GE Healthcare (1 mentions)
Lightcycler, by Roche (1 mentions)
Sybr green pcr master mix, by Roche (1 mentions)
3 protocols using horseradish peroxidase hrp conjugated goat anti rabbit antibody
1
Western Blot Protein Quantification
2
AGE-Induced Mitochondrial Dysfunction Study
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pitavastatin was provided by Huawei Pharmaceutical Technology Development Co., (Fuzhou,China). AGE-modified bovine serum albumin (AGE-BSA) was purchased from Calbiochem (catolog number:121800, San Diego, USA). RAGE antibody was purchased from R&D (catolog number: AF1616, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were provided by Gibco (NY, USA). Collagenase II was purchased from Sigma (Santa Clara, USA). p62, beclin1, and GAPDH antibodies were obtained from CST (#5114, #3738, #2118, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody was purchased from Abcam (6721, USA). Mitochondrial membrane potential assay kit with JC-1 and Reactive Oxygen Species assay kit were from Beyotime (Shanghai, China).
3
Quantifying SATB1 mRNA and Protein Expression in Gastric Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
With TRIzol reagent, the RNA of the gastric cancer cells was extracted. With the Reverse
Transcription System kit, the reverse transcription was done to provide the first-strand
cDNA. After transcription, using the SYBR™ Green PCR Master Mix, PCR was taken
with a Roche Light Cycler (Hoffman-La Roche Ltd., Basel, Switzerland), and a PCR procedure
described as follows: denaturation step: 95°C for 2 minutes; 40 cycles: 3 seconds
denaturation at 95°C, 10 seconds annealing at 55°; extension step: 25
seconds at 72°C. By the 2ΔΔCq approach, the
quantification of the expression of mRNA was carried out. The sequences of β-actin
and SATB1 primers are listed inTable S1 .
For the analysis of protein expression by Western blot, the protein was extracted from
the cells and 50 µg protein was first run in SDS-PAGE. After SDS-PAGE running, the
transfer of protein was done by electricity to the membrane made of polyvinylidene
fluoride. After transfer, the antibodies were used to detect the protein: the primary
antibody (polyclonal rabbit anti-human SATB1 antibody; Abcam PLC, Cambridge, MA, USA) and
the secondary antibody (the horseradish peroxidase [HRP]-conjugated goat
anti-rabbit antibody; Abcam PLC). The internal antibody was the β-actin antibody.
The Amersham™ ECL Plus™ kit (GE Healthcare) was used to detect the
bands.
Transcription System kit, the reverse transcription was done to provide the first-strand
cDNA. After transcription, using the SYBR™ Green PCR Master Mix, PCR was taken
with a Roche Light Cycler (Hoffman-La Roche Ltd., Basel, Switzerland), and a PCR procedure
described as follows: denaturation step: 95°C for 2 minutes; 40 cycles: 3 seconds
denaturation at 95°C, 10 seconds annealing at 55°; extension step: 25
seconds at 72°C. By the 2ΔΔCq approach, the
quantification of the expression of mRNA was carried out. The sequences of β-actin
and SATB1 primers are listed in
For the analysis of protein expression by Western blot, the protein was extracted from
the cells and 50 µg protein was first run in SDS-PAGE. After SDS-PAGE running, the
transfer of protein was done by electricity to the membrane made of polyvinylidene
fluoride. After transfer, the antibodies were used to detect the protein: the primary
antibody (polyclonal rabbit anti-human SATB1 antibody; Abcam PLC, Cambridge, MA, USA) and
the secondary antibody (the horseradish peroxidase [HRP]-conjugated goat
anti-rabbit antibody; Abcam PLC). The internal antibody was the β-actin antibody.
The Amersham™ ECL Plus™ kit (GE Healthcare) was used to detect the
bands.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!