Cell lysates were prepared with ice-cold radioimmunoprecipitation assay (RIPA) buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF), and 30 μg of protein was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electro-transfer onto polyvinylidene difluoride (PVDF) membrane (Millipore, USA, #ISEQ00010). The membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline/Tween (TBST), and incubated with a primary antibody overnight at 4 °C followed by a HRP-conjugated secondary antibody (1:1000, Beyotime Biotechnology, China, #A0208) or a HRP-conjugated secondary antibody (1:1000, HuaBio, #HA1006) for 1 h. The bands were visualized with an ECL detection kit (Millipore, #WBKLS0100) and analyzed by the BIO-RAD ChemiDoc XRS system (BIO-RAD, USA). The primary antibodies used in this study are anti-LAMP-1 (1:1000, Cell Signaling Technology, #9091), anti-TRPML1 (1:1000, Atlas Antibodies, #HPA031763), anti-phospho-p70 S6 kinase (1:1000, Cell signaling Technology, #9234), anti-p70 S6 kinase (1:1000, Cell signaling Technology, #9202), anti-TFEB (1:1000, Cell Signaling Technology, #4240), anti-β-actin (1:1000, Cell signaling Technology, #4970) and anti-Vinculin (1:1000, Cell signaling Technology, #4650).
Hrp conjugated secondary antibody
Manufactured by Beyotime
Sourced in China, United States, United Kingdom
HRP-conjugated secondary antibodies are reagents used in immunoassay techniques, such as Western blotting and ELISA. They consist of a secondary antibody that is conjugated to the enzyme horseradish peroxidase (HRP). The HRP label enables the detection and visualization of target proteins or antigens, facilitating quantitative analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (41 mentions)
Ripa lysis buffer, by Beyotime (37 mentions)
Ripa buffer, by Beyotime (26 mentions)
Bca protein assay kit, by Beyotime (23 mentions)
Nitrocellulose membrane, by Merck Group (15 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (15 mentions)
Polyvinylidene fluoride membrane, by Merck Group (13 mentions)
Polyvinylidene difluoride membrane, by Merck Group (10 mentions)
Protease inhibitor cocktail, by Merck Group (8 mentions)
β actin, by Beyotime (8 mentions)
Pvdf membrane, by Bio-Rad (7 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Gapdh, by Proteintech (6 mentions)
Anti gapdh, by Abcam (6 mentions)
Beyoecl plus, by Beyotime (6 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (6 mentions)
β actin, by Cell Signaling Technology (6 mentions)
β actin, by Abcam (6 mentions)
Enhanced chemiluminescence system, by Bio-Rad (5 mentions)
243 protocols using hrp conjugated secondary antibody
1
Western Blot Analysis of Lysosomal Proteins
2
Western Blot Analysis of DCTN4 Protein
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Proteins were extracted from cells using RIPA lysis buffer (Beyotime Biotechnology, Nanjing, China). Equal aliquots of lysates were separated by 8% SDS-PAGE gel and then transferred to PVDF membrane (Millipore, Braunschweig, Germany). Then membrane was blocked with 5% blocking solution for 1.5 h, followed by incubation with DCTN4 or GAPDH antibody (1:1000, Proteintech Group, Inc, Rosemont, IL, USA) overnight at 4°C. Finally, membranes were incubated with HRP-conjugated secondary antibody (1:10,000, Beyotime Biotechnology) for 2 hours. The bands were visualized by ECL detection system (Millipore).
3
Western Blot Analysis of Apoptosis Markers
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HepG2 cells and lysosomes were lysed using RIPA peptide lysis buffer (Beyotime) containing 1% protease inhibitor (Beyotime). The protein content of different fractions was detected via the BCA method. Equivalent amounts of protein (20 μg) were separated on 10% SDS‐PAGE gels, transferred to nitrocellulose membranes and blocked with 1% BSA in TBST for 1 h at 25°C. The membranes were incubated with BAX, Bcl‐2, P53, P62, Cleaved PARP, Cleaved Caspase 3, LC3 and Cathepsin D (Abcam) antibodies overnight at 4°C. After washing, the membranes were incubated with an HRP‐conjugated secondary antibody (1:1000; Beyotime) for 1 h. Images were taken with Tanon‐5200 (Shanghai Tanon Technology Co. Ltd.) for protein level quantification; appropriate film exposures were scanned. The density of bands was determined with ImageJ and normalized to band intensity for GAPDH or β‐Actin.
4
Protein Extraction and Western Blot
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The samples were washed with pre-cooled PBS before being lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing a mixture of protease and phosphatase inhibitors (Pierce, Rockford, IL). The protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Protein samples (25 µg for each lane) were separated using SDS-PAGE gels and transferred to polyvinylidene (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA). Next, the membranes were then blocked with 5% BSA at room temperature for 2 h and incubated with a diluted primary antibody (p-CREB) (phospho Ser133), 1:2500, rabbit monoclonal antibody (Abcam, Cambridge, UK); p-NR2B (phospho Y1472), 1:5000, rabbit polyclonal antibody (Abcam, Cambridge, UK) and for 1 h at room temperature with an HRP-conjugated secondary antibody, 1:20,000 (Beyotime, Shanghai, China). The blots were detected using ECL solution (Pierce, Rockford, IL) and exposed in the Chemiluminescence imaging analysis system (Tanon, Shanghai, China) for 1–10 min.
5
Protein Expression Analysis in Tissues and Cells
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WB analysis was conducted to determine the protein expression in human tissues and colorectal cells. In brief, total protein was extracted, and concentration was measured using the bicinchoninic acid (BCA) protein assay. Total protein (20 μg) was initially separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking in 5% milk, membranes were incubated with specific primary antibodies (1:1,000 dilution) including Cdk5, β-catenin, cyclin D1, c-Myc, cyclin E1, cyclin A, total signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 (p-STAT3), β-actin, GAPDH, and tubulin at 4°C overnight. Membranes were subsequently incubated with HRP-conjugated secondary antibody (Beyotime Institute of Biotechnology). Tubulin and GAPDH served as the loading controls. Levels of protein were quantified using ImageJ 1.43 u/Java 1.6.0–10 from the National Institutes of Health (Bethesda, MD, United States).
6
Western Blot Analysis of Inflammatory Markers
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The levels of the target proteins were determined by performing Western blotting as reported previously [49 (link),50 (link)]. Briefly, compound-treated cell lysates were heated with an SDS loading buffer for 10 min at 95 °C and separated by SDS-PAGE. The proteins were transferred and blotted on a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and incubated with primary antibodies obtained from Beyotime (anti-COX-2, anti-iNOS, anti-p-iκB, anti-iκB, anti-p65, anti-p50, or anti-GAPDH) or obtained from Cell Signaling Technology (anti-p-p65, Beverly, MA, USA) and an HRP-conjugated secondary antibody purchased from Beyotime. The proteins were visualized by chemiluminescence according to the Beyotime ECL kit’s protocol and analyzed using ImageJ 1.53k software.
7
Western Blot Analysis of Splenic NK Cells
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Splenic NK cells from control and HSD-fed mice were sorted by FACS. Total protein was extracted according to the manufacturer's instructions (P0033, Beyotime, Shanghai). Protein extracts were separated by SDS-PAGE on 12% polyacrylamide gels and transferred to a PVDF membrane (3010040001, Sigma, MO, US). After blocking with 5% (w/v) BSA at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibody (anti-gp91phox, EPR6991; anti-p47phox, ab166930; and β-actin, ab8226) (Abcam, Cambridge, US). The following day, the membranes were washed in PBS and incubated for 1 h with an HRP-conjugated secondary antibody (Beyotime, Shanghai, China). Protein bands were detected using an enhanced chemiluminescence kit (Thermo Scientific, Hudson, NH, USA) according to the manufacturer's instructions.
8
Western Blot Analysis of Key Proteins
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After washing twice with ice-cold phosphate-buffered saline (PBS), the cells were incubated at 4 °C with RIPA buffer supplemented with a proteinase inhibitor cocktail (JRDUN biotech, Shanghai, China) for 30 min. Subsequently, the samples were centrifuged at 12,000 r.p.m. for 15 min at 4 °C, and the supernatant was collected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein on the gels were electrophoretically transferred to a nitrocellulose membrane (Millipore, Bredford, MA, USA), and probed with primary antibodies against TRIM65 (Abcam, Cambridge, MA, USA; 1:1000 dilution), TNRC6A (Abcam; 1:1000 dilution), ATG7 (Abcam; 1:200 dilution), LC3 (Abcam; 1:2000 dilution) or cleaved caspase3 (Abcam; 1:1000 dilution). After incubation with an HRP-conjugated secondary antibody (Beyotime, Shanghai, China; 1:1000 dilution), signals were detected using an enhanced chemiluminescence (ECL) detection kit (Millipore). The membrane was probed with a primary antibody against GAPDH (Cell Signaling Technology, Danvers, MA, USA; 1:2000 dilution) for equal loading control.
9
Quantifying Immune Checkpoint Proteins
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To measure the expression of TGFβ RIII, LAG‐3, CPA2, DCN and ANGPTL3, cells were washed with PBS, and total proteins were isolated from cell lines using RIPA buffer (Beyotime). A sample of total proteins (~30 μg) extracted from each cell line was boiled at 100°C for 5 min and then separated using SDS‐PAGE on a 12% polyacrylamide gel. The separated protein bands were transferred onto a PVDF membrane (Millipore) that was subsequently blocked with Quickblock Blocking Buffer (Beyotime; Cat#P0252). The membrane was then incubated overnight with primary antibodies at 4°C. After incubation, the membrane was washed three times with TBST buffer and then incubated with an HRP‐conjugated secondary antibody (1:5000 dilution; Beyotime) for 2 h at room temperature. The membrane was then washed three more times with TBST buffer, and the protein blots were visualized using ECL‐Plus reagent (Millipore). GAPDH was used as a loading control in all western blot studies. The antibodies used for the western blot studies were as follows: anti‐ANGPTL3 (1:1000 dilution; Proteintech; Cat#11964‐1‐AP), anti‐DCN (1:1000 dilution; ABclonal; Cat#A1669), anti‐CPA2 (1:1000 dilution; Santa Cruz; Cat#sc‐515,450), anti‐TGFβ RIII (1:1000 dilution; Santa Cruz; Cat#sc‐74,511), anti‐LAG‐3 (1:1000 dilution; Proteintech; Cat#80867‐1‐RR), and anti‐GAPDH (1:1000 dilution; Proteintech; Cat#60004‐1‐Ig).
10
Western Blot Analysis of ENaC and MAPK Signaling
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The cells were trypsinized, harvested, and lysed, and they were then subjected to protein
extraction. The obtained total proteins were electrophoresed on SDS-PAGE gels and
subsequently transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Skim milk
powder was diluted in TTBS solution. After that, the membranes were blocked with diluted
milk and incubated with primary antibodies against α-ENaC (Proteintech Group, Wuhan,
China; 1:500 dilution), β-ENaC (Proteintech Group; 1:500 dilution), γ-ENaC (Proteintech
Group; 1:1,000 dilution), ERK (Proteintech Group; 1:500 dilution), p-ERK (BIOSS, Beijing,
China; 1:400 dilution), p38 (BIOSS; 1:400 dilution), p-p38 (BIOSS; 1:400 dilution), JNK
(Proteintech Group; 1:1,000 dilution) and p-JNK (Abcam, Cambridge Science Park, Cambridge,
UK ; 1:1,000 dilution) at 4°C overnight. HRP-conjugated secondary antibody (Beyotime
Institute of Biotechnology; 1:5,000 dilution) was added after washing with TTBS. Bands
were subsequently visualized using ECL reagent (Beyotime Institute of Biotechnology). The
optical densities of bands were analyzed by Gel-Pro Analyzer (Media Cybernetics, Inc.,
Bethesda, MD, USA).
extraction. The obtained total proteins were electrophoresed on SDS-PAGE gels and
subsequently transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Skim milk
powder was diluted in TTBS solution. After that, the membranes were blocked with diluted
milk and incubated with primary antibodies against α-ENaC (Proteintech Group, Wuhan,
China; 1:500 dilution), β-ENaC (Proteintech Group; 1:500 dilution), γ-ENaC (Proteintech
Group; 1:1,000 dilution), ERK (Proteintech Group; 1:500 dilution), p-ERK (BIOSS, Beijing,
China; 1:400 dilution), p38 (BIOSS; 1:400 dilution), p-p38 (BIOSS; 1:400 dilution), JNK
(Proteintech Group; 1:1,000 dilution) and p-JNK (Abcam, Cambridge Science Park, Cambridge,
UK ; 1:1,000 dilution) at 4°C overnight. HRP-conjugated secondary antibody (Beyotime
Institute of Biotechnology; 1:5,000 dilution) was added after washing with TTBS. Bands
were subsequently visualized using ECL reagent (Beyotime Institute of Biotechnology). The
optical densities of bands were analyzed by Gel-Pro Analyzer (Media Cybernetics, Inc.,
Bethesda, MD, USA).
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