Western blots was performed as previously described [24 (link)]. Cells were lysed in the RIPA buffer (Beyotime, China), and the protein concentration was measured by using a BCA Protein Assay kit (Pierce, USA). Totally 30 μg cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), blotted onto polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% non-fat dried milk in TBS-T and then probed with primary antibodies. Then the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Boster Bio Tec, China). β-actin was used as a loading control. The primary antibodies included rabbit anti-human EPLIN (1:250, Abcam, USA), mouse anti-β-actin (1:1000, Sigma, USA). Membranes were then detected by using the enhanced chemoluminescence (ECL) system (Clinx Science Instruments, China).
Ecl system
Manufactured by Clinx
Sourced in China
The ECL system is a laboratory equipment used to detect and measure the presence of specific proteins in a sample. It utilizes chemiluminescent detection technology to generate a luminescent signal that can be quantified and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Bca assay, by Beyotime (3 mentions)
Image lab version 3, by Bio-Rad (3 mentions)
Ab32042, by Abcam (3 mentions)
Ab32351, by Abcam (3 mentions)
Goat anti rabbit igg hrp se134, by Solarbio (3 mentions)
Goat anti mouse igg hrp se131, by Solarbio (3 mentions)
Beyogel plus page, by Beyotime (3 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Abcam (2 mentions)
Caspase 3, by Abcam (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Calreticulin, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Lamin a c, by Abcam (1 mentions)
18 protocols using ecl system
1
Quantitative Western Blot Analysis of EPLIN Protein
2
Comprehensive EV Protein Profiling Protocol
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Cells and EVs were lysed with working solution of the RIPA buffer (Beyotime) containing a protease inhibitor cocktail (Roche). Proteins were separated by sodium dodecyl sulfate–12% polyacrylamide gel (SDS–PAGE) electrophoresis and blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were incubated with PBST (PBS and 0.1% Tween 20) containing 5% skim milk for 1 h, and successively incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. Membranes were developed using an enhanced chemoluminescence (ECL) system (Clinx Science Instruments, Shanghai, China). The primary and secondary antibodies included β-actin (1:5000, Genetex), CD63, CD9, Alix, voltage-dependent anion-selective channel (VDAC) 137 (link), Calreticulin, Lamin A/C, Tau, Map-2 (1:1000, Abcam, Cambridge, UK), Cleaved caspase-3 (aCasp3), Cav-1 (1:1000, CST, Boston, MA), HRP-conjugated goat anti-rabbit IgG (Genshare, Xian, China) and HRP-conjugated goat anti-mouse IgG (HRP) (Genshare).
3
Characterizing SARS-CoV-2 Spike Protein
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The expression of mRNA-LNPs was analyzed by western blot analysis. Whole-cell lysates and supernatants were combined with a loading buffer containing dithiothreitol, separated by 10% SDS-PAGE, and transferred onto a polyvinylidene difluoride (PVDF) membrane using a semidry blotting apparatus (15 V and 60 min). The membrane was blocked with nonfat milk in a PBS buffer containing 0.5% Tween-20. Samples were incubated with a primary antibody (SARS-CoV-2 S1 polyclonal antibody at 0.1 µg/mL, CanSino Biologics, China) for 1.5 h followed by incubation with a secondary goat anti-rabbit IgG-HRP for 45 min. The membrane was incubated with a western blot substrate (Solarbio, China), and images were acquired using an ECL system (CLINX, China).
4
Western Blot Analysis of JEV Envelope Protein
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Purified recombinant Env protein was first separated by SDS-PAGE, and then electro-transferred to polyvinylidene difluoride (PVDF) membrane using a semi-dry blotting apparatus (400 mA, 40 min). After transfer, the membrane was blocked with 5% skim milk in TBS buffer containing 0.1% Tween-20 for 2 h at 37 °C, rinsed once with PBST. Afterward, the membrane was incubated with primary antibody (mouse polyclonal antibodies against JEV Env at 0.1 µg/mL, prepared in blocking buffer). After 1 h incubation, the membrane was washed five times with TBS containing 0.1% Tween-20 and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (0.1 µg/mL prepared in blocking buffer) as the secondary antibody for one hour, and then washed five times. Finally, the membranes were detected by ECL system (CLINX, Shanghai, China).
5
Protein Expression Analysis via SDS-PAGE
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Cells were lysed in the RIPA buffer (Beyotime, Shanghai, China), containing 10 mM phenylmethanesulfonyl fluoride (PMSF), and the protein concentration determined using a BCA protein assay kit (Thermo Fisher). To detect the protein level of nuclear p65, the nuclear extraction was prepared using an extraction kit (Beyotime) according to the manufacturer’s instruction. Total cell lysates and nuclear proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes, probed with primary antibodies, followed by HRP-conjugated secondary antibodies. β-actin was used as a loading control. HRP-based detection was done using an enhanced chemiluminescence (ECL) system (Clinx Science Instruments, Shanghai, China). Antibodies are listed in Additional file 2 : Table S3.
6
VISTA Protein Expression Analysis by Western Blot
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RIPA lysis buffer (Epizyme) was used to lyse cells. After centrifugation, the supernatant was collected in a clean tube. BCA Protein Assay Kit (Epizyme) was applied to quantify protein concentrations. Subsequently, loading buffer (Epizyme) was mixed with supernatant and heated. Samples of equal amounts were separated by 10% PAGE Gel Fast Preparation Kit (Epizyme) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). PVDF membranes were blocked with skimmed milk powder (Servicebio) dissolved in TBST at room temperature for 2 h. The membranes were incubated with primary antibodies against VISTA (1:1000, CST) and GAPDH (1:10 000, CST; Table S2 ) at 4°C overnight. GAPDH was used as control. After incubation with secondary antibodies (1:5000, Servicebio), the signals were visualised by the ECL system (Clinx Science Instruments). Experiments were performed in triplicate.
7
Western Blot Analysis of Nuclear p65
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HUVECs or retinal vasculature were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) containing phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich). To detect the cytoplasmic and nuclear p65 protein levels, we performed cytoplasmic and nuclear extraction using an extraction kit (Beyotime) according to the manufacturer’s instructions. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with primary antibodies, then horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or horse anti-mouse IgG secondary antibody (1:2,000, Cell Signaling Technology, Boston, MA, USA). Each step was followed by three washes in PBS containing 0.1% Tween 20 for 10 min. Bands were visualized using an enhanced chemiluminescence (ECL) system (Clinx Science Instruments, Shanghai, China). Quantification was performed using ImageJ2x software (Rawak Software, Stuttgart, Germany). The antibodies used are listed in Table S2 .
8
Macrophage Protein Analysis by Western Blot
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Analysis of the proteins extracted from macrophages or ankle joints by Western blot was performed using standard methods. Equivalent amounts of protein from each sample were separated in 10% SDS-polyacrylamide gel and blotted onto a PVDF membrane. The membrane was then blocked with 5% milk (BD, Sparks, MD, United States) and incubated with antibodies against p38, p65, p-p38, p-ERK, p-JNK1/2, p-p65, iNOS, and β-actin (Cell Signaling Technology, Danvers, MA, United States) overnight, and then was hybridized with HRP-conjugated secondary antibody for 1 h. The immunoreactive bands were visualized using a ECL system (CLINX, Shanhai, China). The relative intensities of bands were quantified using Image J.
9
Western Blot Analysis of Osteoblast Proteins
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The total protein of primary osteoblasts was extracted using RIPA lysis buffer (Beyotime, China) and quantified by BCA assay (Beyotime, China). Equal amounts of proteins (100 μg) were separated via BeyoGel™ Plus PAGE (Beyotime, China) and then transferred to a PVDF membrane (Millipore, USA). After transferring, the membranes were blocked with 5% fat-free milk for 1 h. The membranes were incubated with primary antibodies (Bax, ab32503; Caspase-3, ab32351; and cleaved-Caspase-3, ab32042) purchased from Abcam (USA) and GAPDH, 10494-I-AP purchased from Proteintech (China) at 4°C overnight. The membranes were incubated with second antibodies (goat anti rabbit IgG HRP SE134, goat anti mouse IgG HRP SE131, Solarbio, China) at 37°C for 1 h. The ECL system (CLINX, China) was used for exposing protein bands. The intensity of the bands was analyzed using Image Lab (version 3.0, Bio-Rad, USA).
10
Chondrocyte Protein Expression Analysis
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Chondrocytes were treated on 6 cm dish, washed with cold PBS, lysis with RIPA (Solarbio) containing a protease inhibitor mixture.25 A total of 30 μg protein were resolved by 10% SDS‐PAGE, transferred to a PVDF membrane, blocked with 5% milk, incubated with antibodies against COX‐2, iNOS, MMP‐9, MMP‐13, TLR4, TRAF‐6, Syk, p‐Syk, p65, p‐p65 and β‐actin (1:1000; Cell Signaling Technology) at 4°C overnight and hybridized with HRP‐conjugated secondary antibody for 1 hours. The immunoreactive bands were visualized using an ECL system (CLINX). The relative intensities of bands were quantified using Image J.
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