HUVECs were lysed on ice with RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitor cocktails (Roche, Germany). After the cell lysate was centrifuged at 16,000g for 10 min at 4 °C, solution A and solution B were calculated and prepared, and the total protein concentration was determined by the Bio-Rad protein assay. Then the cell lysate was boiled at 105 °C for 10 min to denature the protein. Next, the protein extracts were electrophoresed on an SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane. Then, the membrane was blocked with 5% bovine serum albumin at 26 °C for 1 h, and incubated with each primary antibody overnight against ICAM-1 (Affinity, AF6088), IL-8 (Affinity, DF6998), MCP-1 (Affinity, DF7577), IL-6 (Affinity, DF6087), HIF-1α (Novus, NB100-105), JMJD1A (Proteintech, 12835-1-AP), and β-actin/GAPDH (Proteintech, 66009-1-lg/60004-1-Ig) as the loading control. After adding 5% BSA horseradish peroxidase-conjugated secondary antibodies to the membrane, it was incubated slowly at 25 °C for 120 min, and exposed via enhanced chemiluminescence. Quantification of the intensity of the protein bands were performed with ImageJ.
Df7577
Manufactured by Affinity Biosciences
Sourced in United States
The DF7577 is a compact benchtop centrifuge designed for general laboratory use. It features a maximum speed of 7,500 rpm and can accommodate sample volumes up to 50 mL. The centrifuge is equipped with a brushless motor and digital speed control for precise operation.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Af6088, by Affinity Biosciences (1 mentions)
Df6998, by Affinity Biosciences (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Las4000 analyzer, by GE Healthcare (1 mentions)
Af5198, by Affinity Biosciences (1 mentions)
Af5103, by Affinity Biosciences (1 mentions)
Df6657, by Affinity Biosciences (1 mentions)
Af0639, by Affinity Biosciences (1 mentions)
Af0199, by Affinity Biosciences (1 mentions)
Af5393, by Affinity Biosciences (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab134047, by Abcam (1 mentions)
Ab7028, by Abcam (1 mentions)
Ab32518, by Abcam (1 mentions)
Ab137676, by Abcam (1 mentions)
Ab97726, by Abcam (1 mentions)
3 protocols using df7577
1
Protein Expression Analysis of HUVECs
2
Western Blot Analysis of Inflammatory Markers
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Whole-protein extracts were obtained with sonication and separated on 10% SDS-PAGE and then were transferred onto PVDF membrane (Millipore, USA). Blocked with TBST (pH 7.5) containing 5% BSA, the membranes were incubated within primary antibodies against MCP1 (1 : 1200, DF7577, Affinity), iNOS (1 : 800, AF0199, Affinity), Arg1 (1 : 800, DF6657, Affinity), IL-1β (1 : 800, AF5103, Affinity), phosphorylated NF-κBp65 (1 : 1000, 3039, CST), NF-κBp65 (1 : 1000, 8242, CST), PPARγ (1 : 600, 2435, CST), HO-1 (1 : 1000, AF5393, Affinity), Nrf2 (1 : 1000, AF0639, Affinity), SOD1 (1 : 1000, AF5198, Affinity), the endogenous control β-actin (1 : 1000, 4970, CST), and GAPDH (1 : 1000, 3683, CST) at 4°C overnight. The membranes were then incubated in HRP-conjugated secondary antibodies (1 : 3000, 7074, CST) for 1 h at room temperature. We used the ECL Western Blot Kit (Thermo Scientific Pierce) to examine the bands and scanned them using a LAS4000 analyzer (GE Healthcare). The immunoblot density was examined by ImageJ and normalized by β-actin or GAPDH.
3
Protein Expression Analysis via RIPA Extraction
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Protein extraction was performed using the protease inhibitor-contained radioimmunoprecipitation assay (RIPA) buffer (R0010; Solarbio, Beijing, China), and the concentration was measured using the BCA protein assay kit (Thermo Fisher Scientific). The protein sample was separated using freshly prepared SDS-PAGE, electrotransferred onto polyvinylidene fluoride (PVDF) membranes, and then probed with primary antibodies. Next, the membrane was re-probed with goat anti-rabbit IgG (1:10,000, ab6721; Abcam), and the immunoblots were visualized with enhanced chemiluminescence detection reagents. Additionally, the gray values of the target protein bands were quantified by employing the Image Pro Plus 6.0 software (Media Cybernetics, USA), with GAPDH used for normalization. The primary antibodies used were the following: anti-HDAC1 antibody (1:2,000, ab7028; Abcam), anti-KLF5 antibody (1:2,000, ab137676; Abcam), anti-IKBα antibody (1:1,000, ab32518; Abcam), anti-p65 antibody (1:1,000, ab16502; Abcam), anti-p-p65 antibody (1:1,000, ab97726; Abcam), anti-VCAM-1 antibody (1:2,000, ab134047; Abcam), anti-ICAM-1 antibody (1:1,000, ab7815; Abcam), and anti-MCP-1 antibody (1:1,000, DF7577; Affinity Biosciences, USA). All western blot tests were conducted with technological triplicate to obtain sufficient data for further analysis.
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