Basement membrane proteins were extracted by centrifugation and stored in liquid nitrogen. The protein concentration was measured using a BCA protein analysis kit (Pierce Company, Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. The samples were boiled for 7 minutes and then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene difluoride membranes (Thermo Fisher Scientific, Waltham, MA, USA) and blocked with TBST (NaCl 500 mM, Tris 20 mM, pH 7.5) containing 5% skim milk for 1 hour, and then incubated with primary antibodies (rabbit connexin 26 and connexin 30 polyclonal antibodies, 1:1000; Invitrogen; mouse β-actin monoclonal antibody, 1:2000; ZSGB-BIO) overnight at 4°C. The membranes were then washed with TBST for 10 minutes (total four times) and incubated with HRP-labeled secondary antibody (1:5000; ZSGB-BIO) for 1 hour at room temperature. The membranes were washed three times with TBST for 5 minutes each after each incubation. Finally, the membranes were treated with enhanced chemiluminescence color solution (Beyotime, Shanghai, China) and scanned using image analysis software (BandScan 5.0) for semi quantitative analysis.
Hrp labeled secondary antibody
Manufactured by ZSGB-BIO
Sourced in China
The HRP-labeled secondary antibody is a laboratory reagent used in immunoassays and other immunochemical techniques. It consists of a secondary antibody molecule conjugated with the enzyme Horseradish Peroxidase (HRP). The HRP label allows for the detection and quantification of target analytes through a colorimetric or chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Bnip3, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine solution, by ZSGB-BIO (1 mentions)
Horseradish peroxidase, by ZSGB-BIO (1 mentions)
Dp controller, by Olympus (1 mentions)
Mouse anti gapdh, by ZSGB-BIO (1 mentions)
Palmitoleic acid, by Merck Group (1 mentions)
P0500, by Merck Group (1 mentions)
O1008, by Merck Group (1 mentions)
P9417, by Merck Group (1 mentions)
Stearic acid, by Merck Group (1 mentions)
Oleic acid, by Merck Group (1 mentions)
S4751, by Merck Group (1 mentions)
Rabbit anti gli1, by Cell Signaling Technology (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Anti cdk1, by Abcam (1 mentions)
Ripa extraction reagent, by Solarbio (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
16 protocols using hrp labeled secondary antibody
1
Basement Membrane Protein Extraction and Analysis
2
Immunohistochemical Analysis of SHMT2
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The expression and location of SHMT2 were estimated with IHC by the streptavidin peroxidase complex method according to the method described in a previous study [12 (link)]. In brief, after being deparaffinized and rehydrated with xylene and graded alcohol, tissues were incubated in boiled 0.01 M citrate buffer (pH=6.0) for the best antigen retrieval. We used 3% H2O2 to inactivate the endogenous peroxidase. Following the blockage of unspecific binding by 5% bovine serum albumin (BSA), tissues were incubated in primary antibody of SHMT2 at 1: 100 (Abcam, Cambridge, MA, USA, cat. no. EPR3198) at 1: 100 dilution at 4°C overnight. After rinsing in phosphate-buffered saline 3 times, tissues were incubated in HRP-labeled secondary antibody (ZSBio, Beijing, China) at room temperature for 30 min. Finally, the complex reagent of streptavidin peroxidase (ZSBio, Beijing, China) was used, and 3,3′-diaminobenzidine solution (ZSBio, Beijing, China) was applied for final visualization of the antigen.
3
Immunohistochemical Staining of Colorectal Tumors
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The tumors were harvested, formalin-fixed, paraffin-embedded, and made into slides. Then, the slides were deparaffinized in xylene and then were rehydrated through an ethanol gradient. The sections were heated at 100°C in 0.01 mol/L sodium citrate buffer (pH 6.0) for 10 min for antigen retrieval. The slides were inactivated with 3% H2O2 for 15 min at room temperature in the dark place, blocked with 10% goat serum for 10 min at 37°C, and incubated with 100 µl primary antibodies diluted in PBS to each section overnight at 4°C, 100% humidity. Next day, the sections were stained with horseradish peroxidase (HRP)–labeled secondary antibody (ZSGB-BIO, Beijing, China) at 37°C for 40 min. Finally, the sections were incubated with 3,3′-diaminobenzidine (DAB, ZSGB-BIO, Beijing, China, and ZLI-9019) to perform color development. After counterstaining with hematoxylin, the sections were observed and recorded using a microscope with an Olympus DP controller (Olympus). A staining score was calculated by multiplying two scores of staining intensity (0: negative staining; 1: weak staining; 2: moderate staining; 3: strong staining) and intensity (0: 0–25%; 1: 25–50%; 2: 50–75%; 3: >75%) of positive stained colorectal cancer cells.
4
Immunohistochemical Staining of Fixed Tumor Samples
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The fixed tumors were dehydrated, embedded in paraffin, and then sliced into 4 µm thick slices, deparaffinized with xylene and rehydrated with different concentrations of ethanol. Next, slices were heated in 0.01 mol/L sodium citrate buffer at 100 °C for 10 min to perform antigen retrieval. After inactivation of endogenous peroxidase by 3% H2O2, the slices were blocked with 10% goat serum and incubated with 50 µL diluted primary antibodies overnight at 4 °C. The Next day, slices were stained with HRP-labeled secondary antibody (ZSGB-BIO, Beijing, China) at 37 °C for 30 min. Finally, the sections were incubated with DAB for 3 min for color development. After counterstained with hematoxylin, all sections were observed and recorded by a microscope. A staining score was calculated by the staining intensity score (negative, weak, moderate, and strong scored as 0, 1, 2, and 3, respectively) multiplying the score of positively stained cell numbers (0, none; 1, 1–40%; 2, 40–70%; 3, 70–100%) [30] (link).
5
Quantification of MAPK8IP1 and SH3GLB1 Levels
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Western Blot (WB) analysis was performed as previously described [51]. Mouse anti-GAPDH (1:1000, ZSGB-BIO), Rabbit anti-MAPK8IP1 (1:200, Proteintech), Rabbit anti-SH3GLB1 (1:500, Proteintech), and HRP-labeled secondary antibody (1:3000, ZSGB-BIO) were used to quantify MAPK8IP1, SH3GLB1 and GAPDH (control) levels.
6
CD36 Binding Assay for Lipid Molecules
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OxLig-1 was synthesized and identified by our research group according
to previous methods.33 (link),53 (link) Oxlig-1, oleic acid (O1008, Sigma),
palmitoleic acid (P9417, Sigma), stearic acid (S4751, Sigma), and
palmitic acid (P0500, Sigma) were dissolved in anhydrous ethanol to
a final concentration of 50 μg/mL and coated into 96-well polystyrene
plates (50 μL/well) by ethanol evaporation. The plates were
blocked with PBS containing 1% gelatin for 1 h. Then, recombinant
human CD36 protein with His tag (50 μg/mL, 10752H08H50, Invitrogen),
anti-His antibody (1:1000, ZSGB-BIO, China), and HRP-labeled secondary
antibody (1:1000, ZSGB-BIO, China) was added and incubated for 1 h,
Finally, the color was developed with o-phenylenediamine
buffer containing H2O2 and terminated by 2 N
H2SO4. Absorbance was measured at 492 nm. In
each step, the wells were extensively washed with PBS containing 0.05%
Tween-20.
to previous methods.33 (link),53 (link) Oxlig-1, oleic acid (O1008, Sigma),
palmitoleic acid (P9417, Sigma), stearic acid (S4751, Sigma), and
palmitic acid (P0500, Sigma) were dissolved in anhydrous ethanol to
a final concentration of 50 μg/mL and coated into 96-well polystyrene
plates (50 μL/well) by ethanol evaporation. The plates were
blocked with PBS containing 1% gelatin for 1 h. Then, recombinant
human CD36 protein with His tag (50 μg/mL, 10752H08H50, Invitrogen),
anti-His antibody (1:1000, ZSGB-BIO, China), and HRP-labeled secondary
antibody (1:1000, ZSGB-BIO, China) was added and incubated for 1 h,
Finally, the color was developed with o-phenylenediamine
buffer containing H2O2 and terminated by 2 N
H2SO4. Absorbance was measured at 492 nm. In
each step, the wells were extensively washed with PBS containing 0.05%
Tween-20.
7
Comprehensive Protein Detection Assays
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Western Blot (WB), immunofluorescence (IF) and immunohistochemistry (IHC) assays were performed as previously described [49 (link), 50 (link)]. Rabbit anti-Sufu (1:1000, Cell Signaling Technology), rabbit anti-Gli1 (1:1000, Cell Signaling Technology), rabbit anti-fibrillarin (1:1000, Proteintech) mouse anti-β-actin, anti-GAPDH antibody (1:1000, Sigma-Aldridge) and HRP-labeled secondary antibody (1:4000, Zsbio) were used in WB. Rabbit antibodies against Sufu, Gli1 (1:100, Bioss) and TRITIC labeled secondary antibody (1:100, Zsbio) were used for IHC and IF.
For co-Immunoprecipitation (co-IP), the cells were lysed using RIPA buffer and incubated with 20 μL of protein-A/G PLUS-Agarose beads (Santa Cruz) and 1 μg of the appropriate primary antibodies at 4°C overnight. After washing 3 times with RIPA, the samples were analyzed through WB.
For co-Immunoprecipitation (co-IP), the cells were lysed using RIPA buffer and incubated with 20 μL of protein-A/G PLUS-Agarose beads (Santa Cruz) and 1 μg of the appropriate primary antibodies at 4°C overnight. After washing 3 times with RIPA, the samples were analyzed through WB.
8
Western Blot Analysis of Protein Expression
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Total proteins were extracted using RIPA extraction reagents (Solarbio, Beijing, China), and the protein concentration was checked by BCA Protein Assay Kit (Beyotime, Shanghai, China). A total of 50 μg of protein was separated by 10% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% BSA for 1 hr at room temperature, incubated with the primary antibody at 4°C overnight, and subsequently incubated with horseradish peroxidase (HRP)-labeled secondary antibody (ZSGB-Bio, Beijing, China, 1: 5,000) for 1 hr at room temperature before detection by ECL (Thermo, Waltham, MA, USA). The information of antibodies was as follows: anti-Sp1 (Santa Cruz, Dallas, TX, USA, 1:1,000), anti-CDK1 (Abcam, Cambridge, UK, 1:1,000), and anti-GAPDH (Abcam, 1:1,000) .
9
Protein Extraction and Western Blotting Analysis
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Total proteins were extracted from model mouse lung tissue and cells using a prepared SDS lysis buffer for Western blotting analysis. Then, 60 ug of protein was isolated using 12% SDS-polyacrylamide gel and imprinted onto polyvinylidene fluoride membrane (PVDF, Merck Millipore, Germany). Then, the membrane was sealed in 5% skim milk for 1.5 h. β-actin (Abcam, # ab8226), Collagen I (Arigo Biolaboratories, #ARG21965), HIF-1a (Proteintech, #20960-1-AP), LC3A/B (Cell signaling technology, #12741), P62 (Cell signaling technology, #16177S), Phospho-SQSTM1/p62(Ser349) (CST, #E7M1A), Bcl-2 (Abcam, #ab182858), BNIP3 (Santa Cruz Biotechnology, #sc-56167), BNIP3L (Proteintech, #12986-1-AP), BECN1 (Boster, #PB0014), and SOD2 (Boster, #BA4566) were used. The PVDF membranes were subjected to incubation with antibodies above at 4 °C overnight, then HRP-labeled secondary antibody (ZSGB-BIO, Beijing, China) was added and incubated for 1 h, and the immune reaction was detected by the ECL luminescence system.
10
Screening Phage Binding to CD44v3-v10 Cells
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HEK-293 cells were seeded in 96-well plates and transfected with CD44v3–v10 plasmid and empty plasmid separately. 48h after transfection, the candidate phages and the unrelated phages (URps) were added (1×1010 pfu/well) and incubated with cells at 37 °C for 1 h. After six times wash, bound phages were probed by a goat anti-M13 major coat protein antibody (1:200 v/v; Santa Cruz), which subsequently incubated with HRP-labeled secondary antibody (1:5,000 v/v; ZSGB-BIO). A color-developing reaction was performed with TMB kit (Bioss, Beijing, China). Optical density of 450 nm (OD450) was taken by a microplate reader (Biotek, Winooski, VT, USA). Selectivity rate was determined with the formula: (ODpv – ODbv)/(ODpc – ODbc). ODpv and ODpc represented OD450 of the wells where phages incubated with CD44v3–v10 over expressed cells and control cells. ODbv and ODbc indicated background OD450 of two kinds of cells without incubation with phages.
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