Total proteins were extracted using a protein extraction kit (Phygene). A BCA Protein Assay Kit (Phygene) was used to quantify the concentration of protein. After proteins were denatured at 95°C for 10 min, a Tricine‐SDS‐PAGE Gel Kit (Phygene) was used to prepare the SDS‐PAGE gels separating protein bands. The proteins were transferred onto nitrocellulose membranes (Roche) and immersed in 5% defatted dry milk to block aspecific signals. The membranes were incubated with anti‐matrix metalloprotein 2 (anti‐MMP2, 1:2000; Cusabio Biotech), anti‐MMP9 (1:800; Cusabio Biotech), anti‐RAB3D (1:1000; Affinity), and anti‐β‐actin (1:5000; Affinity). Subsequently, the membranes were washed using Tris buffered saline Tween (Millipore) and incubated with horseradish peroxidase‐labeled secondary antibody (1:5000; Affinity). Biuret Reagent (A + B) (Phygene) was used to develop protein blots. β‐actin served as a control.
Horseradish peroxidase labeled secondary antibody
Manufactured by Affinity Biosciences
Sourced in China
Horseradish peroxidase‐labeled secondary antibody is a type of enzyme-labeled antibody used in various immunoassay techniques. It consists of a secondary antibody that is conjugated with the enzyme horseradish peroxidase. This enzyme-labeled antibody is commonly used as a detection reagent in techniques such as Western blotting, ELISA, and immunohistochemistry to amplify and visualize the signal from the primary antibody-antigen interaction.
Automatically generated - may contain errors
Lab products found in correlation
Tris buffered saline tween, by Merck Group (1 mentions)
Nitrocellulose membrane, by Roche (1 mentions)
Bca protein assay kit, by Phygene (1 mentions)
Anti rab3d, by Affinity Biosciences (1 mentions)
Anti β actin, by Affinity Biosciences (1 mentions)
Enhanced chemiluminescence substrate, by Merck Group (1 mentions)
Ab75975, by Abcam (1 mentions)
Ab7028, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
2 protocols using horseradish peroxidase labeled secondary antibody
1
Protein Expression Analysis by Western Blot
2
Western Blot and Immunoprecipitation Analysis
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After transfection, cell extracts were harvested using lysis buffer (Beyotime, China).
For Western blot analysis, the proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, United States). The membranes were blocked and incubated with the primary antibodies of MCM5 (ab75975, Abcam, United Kingdom, 1:1000), HDAC1 (ab7028, Abcam, United Kingdom, 1:500), E-cadherin (3195, CST, United States), Vimentin (5741, CST, United States), MMP2 (40994, CST, United States), MMP9 (13667, CST, United States), and GAPDH (ab8245, Abcam, United Kingdom, 1:2000) at 4°C. GAPDH served as the loading control. After 2 h, the membranes were incubated with a horseradish peroxidase-labeled secondary antibody (Affinity, China, 1:2000). Protein expression was visualized using an enhanced chemiluminescence substrate (Millipore, United States). For immunoprecipitation, the cell extracts were centrifuged and incubated with specific primary antibodies at 4°C overnight with constant rotation. After incubation with Protein A agarose beads and washed thrice with lysate, the harvest protein was boiled for 10 min, separated through 10% SDS-PAGE, transferred onto a PVDF membrane, and detected using specific antibodies.
For Western blot analysis, the proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, United States). The membranes were blocked and incubated with the primary antibodies of MCM5 (ab75975, Abcam, United Kingdom, 1:1000), HDAC1 (ab7028, Abcam, United Kingdom, 1:500), E-cadherin (3195, CST, United States), Vimentin (5741, CST, United States), MMP2 (40994, CST, United States), MMP9 (13667, CST, United States), and GAPDH (ab8245, Abcam, United Kingdom, 1:2000) at 4°C. GAPDH served as the loading control. After 2 h, the membranes were incubated with a horseradish peroxidase-labeled secondary antibody (Affinity, China, 1:2000). Protein expression was visualized using an enhanced chemiluminescence substrate (Millipore, United States). For immunoprecipitation, the cell extracts were centrifuged and incubated with specific primary antibodies at 4°C overnight with constant rotation. After incubation with Protein A agarose beads and washed thrice with lysate, the harvest protein was boiled for 10 min, separated through 10% SDS-PAGE, transferred onto a PVDF membrane, and detected using specific antibodies.
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