Binding of Griffithsin to glycans was evaluated by ELISA as previously described. [21 (link), 22 (link)]. Briefly, flat-bottom 96-well microtiter plates (Nunc, Maxiscorp, MD, USA) were coated with 10 µg of ovalbumin (OVA). The plates were then washed with PBS and blocked by 3% (w/v) bovine serum albumin (BSA). Either Griffithsin alone or Griffithsin pre-incubated with 100 mM D-(+)-glucose or D-(+)-mannose was added to the wells. The wells only contained the BSA was as a blank. An anti-His6 antibody (Boster, Wuhan, China) and horseradish peroxidase (HRP)-conjugated secondary antibody (Boster, Wuhan, China) were used to detect Griffithsin binding. After developing with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate, the reaction was stopped with 2 M H2SO4 and plates were read using a microplate spectrophotometer (BioTec) at a wavelength of 450 nm.
Horseradish peroxidase hrp conjugated secondary antibody
Manufactured by Boster Bio
Sourced in China
Horseradish peroxidase (HRP)-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques, such as Western blotting and ELISA. HRP is an enzyme that catalyzes a colorimetric or chemiluminescent reaction, enabling the detection and visualization of target proteins or molecules in a sample.
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Lab products found in correlation
Bca protein assay kit, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Flat bottom 96 well microtiter plates, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence western blotting detection kit, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
P pi3k p85, by Abcam (1 mentions)
P mtor, by Abcam (1 mentions)
P akt t308, by Abcam (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence detection kit, by Roche (1 mentions)
Fluorchem fc2 imaging system, by Bio-Techne (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ecl kit, by Boster Bio (1 mentions)
Occludin, by Abcam (1 mentions)
β actin, by Affinity Biosciences (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
16 protocols using horseradish peroxidase hrp conjugated secondary antibody
1
Griffithsin Glycan Binding Assay
2
Western Blot Protocol for GBP1 and IDO1 Detection
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Western blot was performed as previous described25 (link). Briefly, the cells were lysed for 30 min in RIPA buffer (Santa Cruz Biotechnology, Paso Robles, CA, USA), and centrifuged at 18,000 g for 15 min at 4 °C. 30 μg aliquots of the lysates were separated on a 10% sodiumdodecyl sulfate-polyacrylamidegel. The resolved proteins were then transferred onto nitrocellulose membrane (Bio-Rad, Hercules, California, USA). The membrane was subsequently incubated with primary antibodies followed by a horseradish peroxidase (HRP)-conjugated secondary antibody (Boster, Wuhan, China). Protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Thermo, Canoga Park, CA, USA). Antibodies for western blot were purchased from the following suppliers: rabbit polyclonal anti-human GBP1 antibody (Invitrogen), rabbit polyclonal anti-human IDO1 antibody (Invitrogen).
3
Western Blot Analysis of Trigeminal Nuclei
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The caudal subnucleus of the spinal trigeminal nucleus (Vc) was collected from deeply anesthetized, rapidly decapitated rats at designated time, either proceeded immediately for biochemical studies or kept at −80 °C until use. Western blot was performed as described previously (Xie et al. 2014 (link)). Briefly, 30 μg of protein was separate by SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to PVDF membranes. After blocking in 5 % fat-free milk in Tris-buffered saline containing 0.1 % Tween, immunoblots were probed with antibodies to cleaved synaptosomal-associated protein 25 (cSNAP-25) (1:2000; GeneTex, USA), transient receptor potential ankyrin 1 (TRPA1) (1:3000; Abcame, UK), transient receptor potential vanilloid type 1 (TRPV1) (1:1000, Sigma, USA), transient receptor potential vanilloid type 2 (TRPV2) (1:1500; Sigma, USA) and transient receptor potential vanilloid melastatin 8 (TRPM8) (1:1000; Abcame, UK). The same blots were stripped and reprobed with antibodies to β-actin (1:5000; Santa Crus, USA). The blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000, Boster, Wuhan, China) for 1 h at 37 °C. Immunoreactivity was visualized by chemiluminescence and exposure to a film. Band intensities were quantified by densitometric analysis using a densitometer.
4
Protein Extraction and Western Blotting
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BMDCs were harvested at 24 h after stimulation with LPS and frozen at −80°C. Total protein was extracted using a lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were determined by BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Protein (40 μg/well) was separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA). Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing Tween 20 (TBST) for 2 h at room temperature, then incubated with primary antibodies (p-PI3K p85, p-Akt T308, p-mTOR, Abcam, Cambridge, UK) diluted in a blocking solution (1:1,000) overnight at 4°C, and finally treated with diluted horseradish peroxidase (HRP)-conjugated secondary antibody (1:50,000, BOSTER Biological Technology Co., Wuhan, Chine) at 37°C for 2 h. The immunoreactive bands were analyzed using the BandScan 5.0 (Glyko, Novato, CA, USA) gel imaging software.
5
Western Blot Analysis of Cellular Proteins
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Proteins were separated on a 10% SDS-PAGE gels, transferred to PVDF membrane (polyvinylidenedifluoride, Millipore, USA), and incubated overnight at 4 °C with antibodies directed against SIRT3, SOD2, Akt, phospho(p)-Akt (Ser 473) (1:1,000, Cell Signaling Technology, USA), acetylated(Ac)-SOD2 (Lys68) antibody (1:1,000, Abcam, USA), p-eNOS (Ser 1177), eNOS (1:1,000, BD Biosciences, USA) or β-actin (1:1,000, Santa Cruz, USA). After washing blots to remove excessive primary antibody binding, blots were incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Boster, China) at room temperature. The blots were developed with an enhanced chemiluminescence detection kit (Roche, USA). The immunoblot was visualized with ChemiDocXRS (Bio-Rad, Hercules, USA) and the blot densities were analyzed with Lab Image software.
6
Assessing CHST12 Expression in GBM
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The samples of GBM tissues and adjacent tissues were heated at 60°C for 1 h, dewaxed using xylene, and rehydrated using descending concentrations of alcohol. Then, the tissue samples were microwaved with ethylenediaminetetraacetic acid for 30 min to retrieve the antigen. To inhibit endogenous peroxidase and decrease nonspecific binding, 0.3% H2O2 and 5% BSA were used. The samples were then incubated with anti-CHST12 antibodies (dilution 1:100; catalog number: 15,341-1-AP; Proteintech, Wuhan, China) overnight at 4°C. After washing three times with PBS, the samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (dilution 1:100; catalog number: BM3894; Boster, Wuhan, China) for 2 h. Finally, the samples were counterstained with hematoxylin and incubated with diaminobenzidine. The expression level of CHST12 in tissues was determined using the product of the depth of staining (0, no; 1, slight stain; 2, moderate stain; and 4, depth stain) and number of stained cells (0, <1%; 1, 1%–33%; 2, 34%–66%; and 3, >66%).
7
Immunoblotting for PRRSV and FljB Detection
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The antisera against PRRSV or FljB were generated by immunizing mice with the inactivated PRRSV strain HH08 or FljB protein mixed with Freund's adjuvant. The purified recombinant proteins of rGP5 and rGP5-FljB were both subjected to SDS-PAGE and then transferred to a nitrocellulose membrane. The membranes were blocked with blocking buffer (5 % non-fat dry milk and 0.05 % Tween-20 in phosphate-buffered saline, PBST) at 4 °C overnight. The next day, the membranes were incubated with a polyclonal antibody against PRRSV or FljB (1:1000 diluted in PBST) at 37 °C for 2 h. After washing three times with PBST, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 diluted in PBST, Boster, China) at 37 °C for 1 h. The protein bands were visualized via a diaminobenzidine enzyme-based color development in the dark that was terminated by distilled water.
8
Western Blot Analysis of YWHAZ Protein
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Clinical specimen was ground into powder in liquid nitrogen and then lysed in lysis buffer to obtain total proteins of each sample. Total proteins in cells were also harvested by using lysis buffer. The concentration of each protein sample was measured using BCA Protein Assay kit (Beyotime, Jiangsu, China). A total of 20 μg of each protein sample was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane for 1 h, and then the membrane was blocked with 5% skimmed milk at room temperature. Human monoclonal primary YWHAZ antibody (1:1000, Santa Cruz, USA) was used to incubate the PVDF membrane overnight at 4°C. The PVDF membrane was further subjected to incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000, Boster, Wuhan, China) for 2 h at room temperature. The blot of YWHAZ protein was detected by the Fluor ChemFC2 Imaging System (Alpha Innotech, SanLeandro, CA, USA). GAPDH was set as the internal control in this research.
9
Quantification of Rab3D Protein Expression
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Radioimmunoprecipitation assay (RIPA) lysis buffer was added into NCI-H1359 and A549 cells to extract total proteins. The total proteins concentration was determined by the BCA kit (Beyotime, Shanghai, China). Each total proteins sample (50 μg) was subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto a PVDF membrane, followed by being blocked with 5% skimmed milk. Subsequently, the membrane was incubated with rabbit anti-RAB3D antibody (1:1000, Abcam, USA) for 12 h at 4°C, followed by being incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, Boster, Wuhan, China) for 2 h at room temperature. The blots were treated by enhanced chemiluminescence and assessed by One software (Bio-Rad, USA). GAPDH was set as the internal control.
10
Western Blot Analysis of Tight Junction Proteins
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20 mg colon tissue was lysed with 200 μL radioimmunoprecipitation (RIPA) lysis solution. The subsequent protocols were performed as previously described [30 (link)]. Briefly, total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA). Membranes were blocked with 5% skim milk and incubated with primary antibodies against zonula occludens-1 (ZO-1, Abcam, USA), occludin (Abcam, USA), and β-actin (Affinity, USA) overnight. Then, horseradish peroxidase (HRP)-conjugated secondary antibodies (Boster, China) were used to incubate the membranes for 2 h. After that, the membranes were visualized with an ECL kit (Boster, China).
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