The frozen wing samples were ground and transferred to centrifuge tubes with RIPA/PMSF (Solarbio, China). The tubes with comminuted samples were incubated in ice for 2 h. The whole divided protein was collected by centrifuging at 4°C for 10 min (12000×g) and adjusted to almost the same protein level. Soluble protein with 4× sample loading buffer was separated on 8–12% SDS-PAGE gel and electrophoresed at 4°C. The protein was transferred onto NC membrane (Millipore Corporation, Billerica, MA, USA) by wet transfer after electrophoresis, then the NC membrane was blocked with 5% fat-free milk in TBST. The MC1R antibody (ab 180776, Abcam, UK), ASIP antibody (ab151033, Abcam, UK), and β-actin antibody (ab8227, Abcam) were incubated at 4°C overnight. The membrane was washed 3 times using TBST buffer, then hybridized with HRP-conjugated goat anti-rabbit IgG for 2 h at room temperature. After washing 5 times with 1× TBST, the proteins of MCIR, ASIP, and β-actin were detected on the membrane with the ultra-sensitive horseradish from the catalase DAB color kit (Sangon Biotech, China). The bands on the membrane were scanned by a densitometric analysis system (Bio-Rad).
Densitometric analysis system
Manufactured by Bio-Rad
The Densitometric analysis system is a laboratory equipment designed to quantify and analyze the intensity of bands or spots on electrophoretic gels or blots. It can measure the optical density or relative intensity of these bands, providing a quantitative assessment of the samples under investigation.
Automatically generated - may contain errors
Lab products found in correlation
Ab180776, by Abcam (1 mentions)
Ripa pmsf, by Solarbio (1 mentions)
Nc membrane, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
Extraction buffer, by Beyotime (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Ecl detection kit, by Beyotime (1 mentions)
Ab106230, by Abcam (1 mentions)
2 protocols using densitometric analysis system
1
Protein Expression Analysis of MC1R and ASIP
2
Western Blot Analysis of CIRP Protein
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Briefly, the tissues were washed 3 times in ice-cold phosphate-buffered saline (PBS), and the total proteins were extracted with in extraction buffer (Beyotime, China). It was separated on 10% SDS-PAGE gel using a Bio-Rad apparatus and then transferred electrophoretically onto enhanced chemiluminescence (ECL) polyvinylidene fluoride (PVDF) membranes (Amersham, USA) using a Bio-Rad Mini Trans-Blot Cell (Bio-Rad, Hercules, CA, USA). The membranes were then blocked with 5% nonfat dry milk and 0.1% Tween 20 in Tris-buffered saline and subsequently incubated with Anti-CIRP (ab106230, Abcam; dilution 1:1000 in PBS) in Tris-buffered saline at 37°C for 2 h, which contained 5% non-fat dry milk and 0.1% Tween 20. Then, the membrane was washed 3 times in Tris-buffered saline, each time for 10 min. The horseradish peroxidase conjugated mouse antigoat IgG (sc-2354, Santa Cruz; dilution 1:8000 in PBS containing 1% bovine serum albumin (BSA) was used to detect conjugates and the antibody-antigen complexes. The membrane was washed 3 times in Tris-buffered saline, each time for 10 min, followed by use of an ECL detection kit (Beyotime, China). Intensity of the bands on the blots was measured by a densitometric analysis system (Bio-Rad). Intensity of b-actin bands were used for normalisation.
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