Tissue samples were stored at ‒80 °C and homogenized in NETT buffer (150 mM NaCl + 5 mM EDTA + 10 mM Tris + 1% TritonX-100 in deionized H2O) with protease inhibitor. Small intestine samples were divided into six equal sections, and sections corresponding to the jejunum were used for lysis. Unfortunately, some small intestine samples degraded during lysis, so only undegraded samples were used for western blot analysis. Protein concentrations of the collected lysates were analyzed with the RC DCTM Protein Assay (Bio-Rad Life Science, Hercules, CA, USA). 100–120 μg of protein from tissue lysates were separated by gel electrophoresis in a 10% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes (GVS, Sanford, ME, USA). Proteins were probed using our custom rabbit anti-mouse primary antibodies, a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (NA9340; GE Healthcare, Chicago, IL, USA), and an HRP-conjugated GAPDH antibody for loading control (HRP-60004; Proteintech, Rosemont, IL, USA). Antibodies were diluted (ZIP14 1:2000, ZnT10 1:1000) in blocking buffer (5% Non-fat dried milk in TBST (Tris-buffered saline + Tween 20)). An enhanced chemiluminescent substrate (SuperSignal West Pico; Thermo Fisher Scientific, Waltham, MA, USA) was used for signal detection with the ChemiDoc™ MP Imaging System (Bio-Rad Life Science).
Hrp 60004
Manufactured by Proteintech
Sourced in United States, China
HRP-60004 is a horseradish peroxidase (HRP) conjugate. HRP is an enzyme that is commonly used as a reporter molecule in various immunoassays and other biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (5 mentions)
Kgp2100, by Keygen Biotech (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Hrp 60008, by Proteintech (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Sa00001 2, by Proteintech (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
70 gar0072, by MultiSciences Biotech (2 mentions)
Imagequant las4000 mini image analyzer, by GE Healthcare (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Ab125066, by Abcam (2 mentions)
Hrp 66240, by Proteintech (2 mentions)
Total protein extraction kit, by Keygen Biotech (2 mentions)
P8340, by Merck Group (2 mentions)
Protease inhibitor, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Et1605 35, by Huabio (2 mentions)
65 protocols using hrp 60004
1
Western Blot Analysis of Intestinal Proteins
2
Mitochondrial Fractionation of SCLC Cells
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Mitochondrial fractionation of NCI‐H1048 or NCI‐H69 monolayer and spheroid cells were extracted by using Mitochondria/Cytosol Fractionation Kit (ab65320, Abcam, Cambridge, MA, USA) according to the manufacturer's protocol. Briefly, SCLC monolayer cells and spheroid cells were collected and washed with PBS. Cells were then resuspended in cytosol extraction buffer and incubated on ice for 10 minutes, followed by homogenization for non‐mitochondrial fractionation extraction. After centrifugation, mitochondrial components were collected and resuspended in mitochondrial extraction buffer. Both mitochondrial fractionations and non‐mitochondrial fractionations were used for immunoprecipitation analyses. Mitochondrial COX IV (1:1000; ab202554, Abcam) and GAPDH (1:10000; HRP‐60004, Proteintech, Wuhan, China) were used as loading control [37 (link)].
3
Western Blot Analysis of DENV NS3
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Cells were lysed with lysis buffer and subjected to SDS-PAGE. The proteins were transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes (Bio-Rad; 1620177), blocked with 5% skim milk, and incubated at 4°C overnight with anti-DENV NS3 antibody (1:2,000; GeneTex; GTX124252) and horseradish peroxidase (HRP)-conjugated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:5,000; Proteintech; HRP-60004). After three washes with Tris-buffered saline supplemented with 0.1% Tween (TBST) (10 min per wash), goat anti-rabbit IgG (H+L) HRP-conjugated secondary antibody (1:5,000; Beijing Ray Antibody Biotech; RM-3002) was added for 1 h at room temperature. After three washes with TBST (10 min per wash), the NS3 and GAPDH bands were visualized with the Immobilon Western chemiluminescent HRP substrate (Proteintech; PK10001).
4
Western Blot Analysis of AMPK and Apoptosis
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Protein concentrations obtained from the infarcted brain tissues or SH-SY5Y cells were assessed utilizing a BCA Protein Assay Kit (KeyGEN BioTECH, Nanjing, China). Specifically, proteins in identical quantities were treated to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 30 ug proteins added into per lane for the electrophoresis, followed by loading onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, United States). Once the membranes had been blocked, incubation of the membranes using primary antibodies at 4°C throughout the night was performed, followed by another incubation using HRP-conjugated secondary antibodies at ambient temperature for 1 h. The membranes were then exposed and analyzed utilizing an ECL system (Tanon, Shanghai, China). The following were the primary antibodies that were employed in the protocol: anti-AMPK (ab32047), p-AMPK (ab133448), PINK1 (ab186303), Bcl-2 (ab196495), and Bax (ab32503). The secondary antibody used was an HRP-conjugated anti-GAPDH monoclonal antibody (HRP-60004, 1:5000, Proteintech, Rosemont, IL, United States).
5
Salivary Gland Protein Expression Analysis
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Salivary gland tissues were homogenized in cold radioimmunoprecipitation assay lysis (RIPA) buffer, which was mixed with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF). Samples were incubated at room temperature for 30 min, and centrifuged at 12,000 rpm for 15 min at 4 °C. Then The supernatant was collected and boiled at 100 °C for 10 min.
We used 10% SDS PAGE gels to separate equal amounts of proteins, and transferred the proteins using polyvinylidene difluoride membranes. Membranes were blocked with 10% skim milk for 2 h at room temperature. The membranes were then incubated with primary antibodies against NADPH oxidase 4 (NOX4; 1:2000 dilution; 14347-1-AP; Proteintech), Bax (1:5000 dilution; ab3191; Abcam), Bcl-2 (1:1000 dilution; ab182858; Abcam), caspase 3 (1:2000 dilution; ab184787; Abcam), Phospho-p53 (1:2000 dilution; P04637; CST, MA, USA), and GAPDH (1:10,000 dilution; HRP-60004; Proteintech). All blots were detected using ECL chemiluminescence reagents and analyzed using the Image Lab software.
We used 10% SDS PAGE gels to separate equal amounts of proteins, and transferred the proteins using polyvinylidene difluoride membranes. Membranes were blocked with 10% skim milk for 2 h at room temperature. The membranes were then incubated with primary antibodies against NADPH oxidase 4 (NOX4; 1:2000 dilution; 14347-1-AP; Proteintech), Bax (1:5000 dilution; ab3191; Abcam), Bcl-2 (1:1000 dilution; ab182858; Abcam), caspase 3 (1:2000 dilution; ab184787; Abcam), Phospho-p53 (1:2000 dilution; P04637; CST, MA, USA), and GAPDH (1:10,000 dilution; HRP-60004; Proteintech). All blots were detected using ECL chemiluminescence reagents and analyzed using the Image Lab software.
6
Western Blot Characterization of OCC-1 in CRC
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For detection of endogenous OCC-1 polypeptide in CRC cells, western blot was performed according to the previous report in which the polypeptide was identified (31 (link)) using three commercially available primary antibodies (ab83945, ab83948 and ab177759, Abcam) raised against three different regions of human OCC-1 polypeptide.
For detection of other proteins in this study, western blot was performed according to standard methods. In brief, proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes (Bio-Rad) and incubated overnight at 4°C with corresponding antibodies: anti-FLAG M2-HRP (1:2000; A8592, Sigma), anti-GAPDH-HRP (1:30000; HRP-60004, proteintech), anti-ACTB (1:5000; 60008-I-Ig, proteintech), anti-HuR (1:1000; ab136542, Abcam) and anti-β-TrCP1 (1:1000; 1B1D2, 37–3400, Thermo Scientific). A HRP-conjugated sheep anti-mouse IgG secondary antibody (1:5000; SA00001-I, proteintech) was used for the detection of ACTB, endogenous HuR and β-TrCP1. The protein signals were detected using ECL chemiluminiscent substrate (FOREGENE).
For detection of other proteins in this study, western blot was performed according to standard methods. In brief, proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes (Bio-Rad) and incubated overnight at 4°C with corresponding antibodies: anti-FLAG M2-HRP (1:2000; A8592, Sigma), anti-GAPDH-HRP (1:30000; HRP-60004, proteintech), anti-ACTB (1:5000; 60008-I-Ig, proteintech), anti-HuR (1:1000; ab136542, Abcam) and anti-β-TrCP1 (1:1000; 1B1D2, 37–3400, Thermo Scientific). A HRP-conjugated sheep anti-mouse IgG secondary antibody (1:5000; SA00001-I, proteintech) was used for the detection of ACTB, endogenous HuR and β-TrCP1. The protein signals were detected using ECL chemiluminiscent substrate (FOREGENE).
7
Western Blot Analysis of Myogenic and Exosomal Markers
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Cells or EVs were sonicated into the lysis buffer supplemented with phosphatase and protease inhibitors (KGP2100, Keygen Biotech, China). Proteins were transferred onto PVDF membranes. Then the PVDF membranes were blocked by 5% milk (P0216-1500 g, Beyotime, China) and incubated with primary antibodies against GAPDH (1:10000, HRP-60004, proteintech, USA), P16 (1:1000, ab51243, abcam, UK), P53 (1:2000, A10610, ABclonal, China), P21 (1:2000, A1483, ABclonal, China), myogenic differentiation antigen (MyoD) (1:100, ab203383, abcam, UK), myogenin (MyoG) (1:200, ab124800, abcam, UK), myosin heavy chain (MyHC) (1:1000, ab91506, abcam, UK), CD9 (1:1000, ab263019, abcam, UK), CD81 (1:1000, ab109201, abcam, UK), TGS101 (1:1000, ab125011, abcam, UK), and Calnexin (1:1000, ab133615, abcam, UK) at 4 °C overnight. Membranes were then incubated with goat anti-rabbit IgG(H + L) HRP (70-GAR0072, MultiSciences, China) at 37 °C for 1 h. Subsequently, the immune complexes were visualized using a tanon™ high-sig ECL western blotting substrate (180-5001, Tanon, China) and automatic digital gel/chemiluminescence image analysis system (4600SF, Tanon, China).
8
Notch Signaling Regulation by Nanoparticles
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Cells were cultured in a 6-well plate overnight. The following day, nanoparticles loaded with NICD plasmid, nanoparticles loaded with NICD plasmid and conjugated anti-Tie2+Tie1, blank nanoparticles, or cell media were added to the culture. After an additional 24 h with treatment and shear stress, the media was removed, cells washed with 1x PBS, and lysed with radio-immunoprecipitation assay buffer supplemented with protease inhibitor cocktail (Roche). Protein concentrations were determined via the Pierce BCA Assay Kit (ThermoFisher). Antibodies against Notch1 (Invitrogen, MA5-32080), Hey1 (Abnova, H00023462-M02), Hes1 (OriGene, TA400013), and GAPDH (Proteintech, HRP-60004) were probed at suggested dilutions. Secondary antibodies conjugated with horseradish peroxidase were incubated and detected by enhanced chemiluminescence reagent (BioRad). The total protein of each well was measured using ImageJ's Gel Analysis. Similarly, each individual band was measured using the same technique, then normalized to the total protein amount.
9
Quantification of MBP Expression in CC
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Western blot was performed to quantify the expression of MBP. The brain was removed and CC tissue was quickly resected on ice. The CC was ultrasonically homogenized in radioimmunoprecipitation assay lysis buffer (RIPA buffer, Beyotime, Shanghai, China) followed by 12,000 rpm centrifugation for 10 min and the supernatant was collected for western blot analysis. Equal amounts of protein samples (20 μg of total protein) were analyzed by SDS-PAGE with primary antibodies being either rabbit anti-MBP (1:500, Millipore, AB980) or anti-GAPDH (1:10000, Proteintech, HRP-60004). Proteins were detected via incubation with horseradish peroxidase-conjugated secondary antibodies and an ECL chemiluminescence detection system (Tanon, Shanghai, China). The images were obtained using an ImageQuant LAS4000 mini image analyzer (GE Healthcare, Buckinghamshire, UK).
10
Western Blot Protein Expression Analysis
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Western blotting (WB) was carried out routinely. The primary antibodies used here included anti-Oct4 (1:1000, #2750; Cell Signaling), CD144 (1:1000, #2500; Cell Signaling), CD105 (1:1000, ab11414; Abcam), CD31 (1:1000, #3528; Cell Signaling), mouse anti-human GAPDH (1:10000, HRP-60004; proteintech) and mouse anti-human β-actin monoclonal antibody (1:10000, A5316; Sigma). Protein bands were visualized with SuperSignalTM West Pico PLUS Chemiluminescent Substrate (#34580; Thermo).
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