Protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The blots were blocked and incubated with an antibody against KIAA1522 (Sigma), phosphorylated extracellular signal-regulated kinase (pERK) (Thr202/Tyr204), or extracellular signal-regulated kinase (ERK) (CST, Beverly, MI, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Kangcheng, Shanghai, China) was used as a loading control. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (Applygen, Beijing, China). The signals were visualized using a super enhanced chemiluminescence (ECL) detection reagent (Applygen).
Horseradish peroxidase conjugated secondary antibody
Manufactured by Applygen
Sourced in China
Horseradish peroxidase-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA) and Western blotting. It consists of a secondary antibody that is covalently conjugated with the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify the presence of a target protein or antigen in a sample by catalyzing a colorimetric or chemiluminescent reaction when the appropriate substrate is added.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ecl reagent, by Merck Group (2 mentions)
Ripa lysis buffer, by Applygen (2 mentions)
Kiaa1522, by Merck Group (1 mentions)
Enhanced chemiluminescence detection reagent, by Applygen (1 mentions)
Bca assay, by Applygen (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab21685, by Abcam (1 mentions)
Ab11734, by Abcam (1 mentions)
Ab108252, by Abcam (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
β actin, by Applygen (1 mentions)
Myd88, by Cell Signaling Technology (1 mentions)
Tipe2, by Proteintech (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ripa buffer, by Applygen (1 mentions)
7 protocols using horseradish peroxidase conjugated secondary antibody
1
Immunoblotting Analysis of KIAA1522, pERK, and ERK
2
Quantification of Akt and Erk1/2 Phosphorylation in Rat Heart
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Protein was isolated from homogenized heart tissue (n = 4) with nondenaturing lysis buffer added with proteinase inhabitor reagent and the concentrations were calculated by BCA assay(Applygen Technologies Inc., Beijing, China). To determine the phosphorylated level ofAkt(serine[Ser] 473) and Erk1/2 in addition to the total level of the proteins(Cell SignalingTechnology, Beverly, MA), 40 μg homogenized protein was separated on a Bis-Tris Gel and transferred to a nitrocellulose mini membrane (Invitrogen, Carlsbad, CA). Membranes were then probed with a rabbit polyclonal antibody against rat Akt(serine[Ser] 473) and Erk1/2(Cell Signaling Technology, Beverly, MA). The bound primary antibodies were detected with horseradishperoxidase-conjugated secondary antibodies and enhanced chemi- luminescencedetection reagents (Applygen Technologies Inc., Beijing, China). The reported densitometry values were analyzed by Totalab v1.10 software (Totalab Ltd., Newcastle, U.K.) and normalized to total Akt and Erk1/2.
3
Western Blot Analysis of ACE, ACE2, and GRP78
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PVN tissues were homogenized in lysis buffer, and the protein concentration in the supernatant was measured with the BCA protein assay Kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein were separated on 4-15% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked in 5% nonfat dry milk for 1 h and then incubated with primary antibodies to ACE1 (1 : 100, ab11734, Abcam, Cambridge, MA, USA), ACE2 receptor (1 : 500, ab108252, Abcam), GRP78 (1 : 400, ab21685, Abcam), and β-actin (1 : 5000, 66009-1-1 g, Proteintech, Rosemont, IL, USA) overnight at 4°C. After three washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Applygen, Beijing, China) for 1 h at room temperature. The signal was visualized using an enhanced chemiluminescence (ECL) detection system (ImageQuant LAS 4000, GE Co., Boston, MA, USA), and densities of the immunobands were quantitated using Quantity One software (V4.6.2, Bio-Rad Co., Boston, MA, USA). All data were corrected by β-actin.
4
Western Blot and Immunoprecipitation Analysis
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After transfection for 48 h, total cellular protein was extracted with RIPA lysis buffer (Applygen, Beijing). Total protein (40 μg) was separated on an SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, Billerica, MA). After blocking with 5% nonfat milk at room temperature for 1 h, the membrane was probed with primary antibodies (1:1000, Danvers, MA) overnight at 4 °C. The next day, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (1:3000, Applygen, Beijing) at room temperature for 1 h. Proteins were detected by ECL reagents (Millipore, Billerica, MA). For immunoprecipitation, stable PANC-1 transfectants of the pENTER and pENTER LAT2 vectors were seeded in 6-well plates. The cells were then lysed in modified RIPA buffer. The cell lysates were incubated with antibody for 12 h at 4 °C on a rotating plate. The proteins were immunoprecipitated by protein A/G agarose beads (Santa Cruz, USA). The samples were resolved by SDS-PAGE and were subjected to immunoblot analysis.
5
Quantification of Immune Signaling Proteins in Splenic Tissue
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After the protein quantification with BCA kits, the protein levels of TIPE2,
MyD88, TRIF, TRAF3 and TRAF6 in splenic tissue from different groups were
analyzed by SDS-PAGE electrophoresis. The protein was then transferred to
polyvinylidene fluoride membrane after electrophoresis. The membranes were
blocked with 5% skim milk diluted in TBST, followed by an overnight incubation
with primary antibodies against β-actin (1:2000 dilution; Applygen), TIPE2
(1:1000 dilution; protein tech), MyD88 (1:1000 dilution; cell signaling
technology), TRIF (1:1000 dilution; Bioworld), TRAF3 (1:1000 dilution;
Bioworld), TRAF6 (1:1000 dilution; Bioworld). The membranes were subsequently
incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000
dilution; Applygen). Finally, images were examined with an ImageQuant LAS 4000
imager, and strip density was analyzed by Quantity One software.
MyD88, TRIF, TRAF3 and TRAF6 in splenic tissue from different groups were
analyzed by SDS-PAGE electrophoresis. The protein was then transferred to
polyvinylidene fluoride membrane after electrophoresis. The membranes were
blocked with 5% skim milk diluted in TBST, followed by an overnight incubation
with primary antibodies against β-actin (1:2000 dilution; Applygen), TIPE2
(1:1000 dilution; protein tech), MyD88 (1:1000 dilution; cell signaling
technology), TRIF (1:1000 dilution; Bioworld), TRAF3 (1:1000 dilution;
Bioworld), TRAF6 (1:1000 dilution; Bioworld). The membranes were subsequently
incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000
dilution; Applygen). Finally, images were examined with an ImageQuant LAS 4000
imager, and strip density was analyzed by Quantity One software.
6
Western Blot Analysis of Protein Expression
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After 48 hours of transfection in 6-well plates, cells were digested with trypsin solution and lysed with RIPA buffer (Applygen, Beijing). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). After blocking with 5% non-fat dry milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies. The membranes were then washed and incubated with a horseradish peroxidase-conjugated secondary antibody (Applygen, Beijing) at room temperature for 1 hour. Protein bands were visualized with echochemiluminescence (ECL) detection system, and the expression levels of these proteins were evaluated using Image-Pro Plus 6.0 software (Media Cybernetics, USA).
7
Protein Extraction and Western Blot
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After transfection for 48 h, total cellular protein was extracted with RIPA lysis buffer (Applygen, Beijing). Total protein (100 μg) was separated on SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was probed with primary antibodies (1:1000, Danvers, MA) overnight at 4 °C after blocking with 5% non-fat milk at room temperature for 1 hour. The next day, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:3000, Applygen, Beijing) at room temperature for 1 hour. Protein bands were detected by ECL reagents (Millipore, Billerica, MA).
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