Lysates from 40 oocytes in SDS sample buffer were subjected to SDS-PAGE and transferred to membrane. The membranes were incubated in the blocking buffer (0.1% Tween-20, 3% BSA in TBST) at room temperature for 1 hour and then with primary antibodies overnight at 4 °C. After extensive washing in TBST, membranes were incubated with HRP-labeled secondary antibodies (Jackson ImmunoResearch) for 2 hours. The membranes were developed with the ECL Plus Western Blotting Detection kit (GE Healthcare Life Science). Signals were quantified densitometrically using ImageJ software (National Institutes of Health) and expressed as relative values (i.e. normalized to the corresponding β-actin signal of the same membrane). The primary antibodies used for immunoblotting were anti-mCherry (1:500; Abcam), anti-cyclin B1 (1:1000; Cell Signaling), and anti-β-actin (1:2500; Cell Signaling).
Hrp labeled secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
HRP-labeled secondary antibodies are used in immunodetection techniques, such as ELISA and Western blotting, to amplify and visualize target proteins. These antibodies are conjugated with the enzyme Horseradish Peroxidase (HRP), which catalyzes a colorimetric or chemiluminescent reaction, allowing for the detection and quantification of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Bz x710 all in one fluorescence microscope, by Keyence (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl plus western blotting detection kit, by GE Healthcare (1 mentions)
Anti mcherry, by Abcam (1 mentions)
Anti cyclin b1, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Pierce ecl western blotting chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Twist, by Proteintech (1 mentions)
8 br camp, by Enzo Life Sciences (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Mmp13, by Proteintech (1 mentions)
25 protocols using hrp labeled secondary antibody
1
Quantitative Western Blot Analysis
2
ELISA for MUC1 and Survivin Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum from mice was diluted with 1× PBS at a ratio of 1 to 50 to detect antibodies against MUC1 and survivin using ELISA performed according to previously described methods.25 (link) MUC1 and survivin proteins purified from recombinant E. coli BL21 were coated at 100 ng per well overnight. The wells were blocked using BSA, and the cells incubated with primary antibody for 2 h followed by incubation with HRP-labeled secondary antibodies (Jackson ImmunoResearch) for 1 h and then TMB was added. Sulfuric acid (2 M) was used to stop the reaction, and the OD450 values were determined.
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in a buffer containing 50 mM HEPES pH 7.3, 0.15 mM EDTA pH 7.9, 4 mM EGTA, 150 mM NaCl and 1% Triton X-100 supplemented with a cocktail of protease inhibitors (aprotinin, leupeptin, pepstatin-A, and pefabloc, 10 μg/ml) for 30 min at 4 °C. After centrifugation for 15 min at 8000 rpm (4 °C), protein concentration was determined using a BCA kit (Thermo Scientific). Protein levels were normalized to 100 µg per sample and resuspended with 4× laemmli sample buffer (Bio-Rad) before boiling (5 min). Proteins were separated by SDS-PAGE using precast 4–20% gels (Bio-Rad) and then transferred onto a nitrocellulose membrane. Membranes were then blocked with Tris-Tween buffered solution (TTBS; 10 mM Tris, 200 mM NaCl, 0.05% Tween 20, pH 7.4) containing 5% non-fat dry milk for 1 h at RT and then incubated with primary antibodies (rabbit anti-Rnd2, 1/250, Proteintech, 13844–1-AP; mouse anti-beta actin, 1/2000, Sigma, A5316) overnight at 4 °C. HRP-labeled secondary antibodies (Jackson ImmunoResearch) were incubated the following day for 1 h at RT.
4
Epithelial-Mesenchymal Transition Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells from a six-well plate were harvested and lysed using RIPA lysis buffer (Thermo Fisher Scientific, MA, USA). The primary antibodies against E-cadherin (Proteintech, IL, USA), N-cadherin (Proteintech, IL, USA), Vimentin (Proteintech, IL, USA), ZEB1 (Santa Cruz Biotechnology, Dallas, TX, USA), Snail (Santa Cruz Biotechonology, Dallas, TX, USA) and Twist (Proteintech, IL, USA) were used to interact with the targeting protein at 4°C overnight. After washing three times with 1×TBST, the membrane was incubated with HRP-labeled secondary antibodies (Jackson ImmunoResearch, PA, USA) for 1 h at room temperature. The blots were visualized with Pierce ECL Western Blotting chemiluminescence substrate (Thermo Fisher Scientific, MA, USA).
5
MVA Vaccine Immunogenicity Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were intramuscularly (i.m.) injected with MVA vaccine thrice at one-week intervals. 5 × 107 PFUs MVA vaccine was injected per mouse per time (2.5 × 107 PFUs in 50 μL PBS for each limb). The day before each vaccination, blood was drawn from the mice, and serum was prepared.
ELISA was used for the detection of binding antibodies. MVA vectors were coated in the plate at 1 × 106 PFUs per well and was incubated at different dilutions of the serum for 2 h followed by HRP-labeled secondary antibodies (Jackson ImmunoResearch) for 1 h. After reaction with TMB and sulfuric acid (2 M) termination, OD450 values were measured.
Cell viability detection was used for screening the neutralizing antibodies against MVA vector in mouse serum. BHK 21 tk-ts13 cells were plated in 96-well plates at 5000 cells per well and cultured for 24 h. The MVA vectors were incubated with varying dilutions of serum, at 37 C for 30 min, and then were added to the wells. The medium was changed to DMEM with 2% FBS at 2 h post-virus infection, and the cells were cultured for another 48 h. Cell viability was detected using MTT assay. The cell viability ratio (%) was calculated according to the following formula: [(absorbance of experimental group with virus infection− background absorbance)/(absorbance of control group without virus infection − background absorbance)] × 100%.
ELISA was used for the detection of binding antibodies. MVA vectors were coated in the plate at 1 × 106 PFUs per well and was incubated at different dilutions of the serum for 2 h followed by HRP-labeled secondary antibodies (Jackson ImmunoResearch) for 1 h. After reaction with TMB and sulfuric acid (2 M) termination, OD450 values were measured.
Cell viability detection was used for screening the neutralizing antibodies against MVA vector in mouse serum. BHK 21 tk-ts13 cells were plated in 96-well plates at 5000 cells per well and cultured for 24 h. The MVA vectors were incubated with varying dilutions of serum, at 37 C for 30 min, and then were added to the wells. The medium was changed to DMEM with 2% FBS at 2 h post-virus infection, and the cells were cultured for another 48 h. Cell viability was detected using MTT assay. The cell viability ratio (%) was calculated according to the following formula: [(absorbance of experimental group with virus infection− background absorbance)/(absorbance of control group without virus infection − background absorbance)] × 100%.
6
AKT and p-AKT Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies against AKT and p-AKT (Ser473) were purchased from Cell Signaling Technology (Leiden, The Netherlands). Anti-GAPDH was from LabForce (Muttenz, Switzerland). HRP-labeled secondary antibodies were purchased from Jackson ImmunoResearch (Suffolk, UK). Prostaglandins E2 and D2, EP1 agonist (17-Phenyl-trinor-prostaglandin E2), EP3 agonist (Sulprostone), cAMP analogs 8-pCPT-2′-O-Me-cAMP and 8-Br-cAMP, and PKI 14–22 were all purchased from Enzo Life Science (Lausen, Switzerland). The Epac inhibitor (ESI-09) was purchased from Sigma-Aldrich (Buchs, Switzerland).
7
Immunohistochemical Visualization of CCL28
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemistry, human vaginal mucosa sections were used for CCL28 staining. Sections were deparaffinized and rehydrated before the addition of primary antibody anti-human CCL28 for overnight incubation. HRP-labeled secondary antibodies (Jackson Immunoresearch, PA) were used before the addition of substrate DAB. Hematoxylin was used for counterstaining these slides. Subsequently, after thoroughly washing in PBS 3 times slides were mounted with a few drops of mounting solution. Images were captured on the BZ-X710 All-in-One fluorescence microscope (Keyence).
8
Protein Expression Analysis of Chondrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from chondrocytes by incubating them for 5 min at 95° C in lysis buffer [62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 50 Mm DTT, and 0.01% bromophenol blue]. Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto a nitrocellulose (NC) membrane (Bio-Rad, Hercules, CA, USA). The blots were incubated overnight at 4° C with primary antibodies against BMP5 (ABclonal, Woburn, MA, USA), RUNX2 (ABclonal), MMP13 (Proteintech), p16 (Proteintech), p53 (Proteintech), p21 (Proteintech), ERK (Cell Signaling Technology, Danvers, MA, USA), p-ERK (Cell Signaling Technology), p38 (Cell Signaling Technology), and p-p38 (Cell Signaling Technology). Then, the blots were incubated at room temperature for 30 min with the corresponding HRP-labeled secondary antibodies ((Jackson ImmunoResearch Laboratories, Baltimore Pike, USA). The blots were then developed with the Enhanced chemiluminescence kit (Millipore, Burlington, MA, USA) and visualized using the Image J software.
9
Antibody Profiling for MAPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in this study included: anti-phosphoERK1/2 (#4370), anti-phosphoMEK1/2 (#9154), and anti-MEK1/2 (#9124) (Cell Signaling Technology); anti-BRAF (SAB5300503), anti-CRAF (SAB5300393), anti-FLAG (F3165) and anti-β-actin (A2228) (Sigma); anti-HA (MAB6875, Novus Biologicals); anti-ERK2 (sc-154, Santa Cruz Biotechnology); anti-ERK1/2 (A0229, AB clonal); anti-Ki67 (ab16667, Abcam); and HRP-labeled secondary antibodies (Jackson Laboratories). Vemurafenib and LY3009120 were purchased from Medchemexpress; and D-luciferin from Gold Biotechnology. All other chemicals were obtained from Sigma.
10
Immunohistochemical Analysis of CCL28 in Vaginal Mucosa
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemistry, human vaginal mucosa sections were used for CCL28 staining. Sections were deparaffinized and rehydrated before the addition of primary antibody anti-human CCL28 for overnight incubation. HRP-labeled secondary antibodies (Jackson Immunoresearch, PA) were used before the addition of substrate DAB. Hematoxylin was used for counterstaining these slides. Subsequently, after thoroughly washing in PBS 3 times slides were mounted with a few drops of mounting solution. Images were captured on the BZ-X710 All-in-One fluorescence microscope (Keyence).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!