Stomach and spleen were fixed in 4% paraformaldehyde (PFA), embedded in paraffin and stained with hematoxylin and eosin (H&E) according to a standard procedure. For Western blotting, brains and hearts were homogenized in a dounce homogenizer using modified radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA [pH 8.0], 1% NP-40, 0.5% Na deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) supplemented with Complete Protease Inhibitors (Roche Diagnostics) and centrifuged at 12,000g for 12 min. Lysates were subjected to Western blot analysis using standard technologies. The western blots were probed with the primary antibody (mouse monoclonal anti-NDRG4, clone 2G3, Novus Biologicals, T5168), followed by treatment with HRP-conjugated goat anti-mouse IgG (Promega, W4021). Positive signals were visualized using a Pierce ECL western blotting substrate detection Kit (Pierce, 32132). For normalization of signals, mouse anti-α-Tublin (clone B-5-1-2, Sigma, H00065009-M01) monoclonal antibody was used as a loading control.
Hrp conjugated goat anti mouse igg
Manufactured by Promega
Sourced in United States
HRP-conjugated goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is conjugated with horseradish peroxidase (HRP). This reagent can be used in various immunodetection techniques, such as ELISA, western blotting, and immunohistochemistry, to detect and visualize mouse IgG-containing samples.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor, by Roche (1 mentions)
Chemidoc xrs imager, by Bio-Rad (1 mentions)
Hrp conjugated goat anti rabbit igg, by Promega (1 mentions)
Mouse anti histidine tag, by Merck Group (1 mentions)
Sunrise microplate reader, by Tecan (1 mentions)
Mouse fh, by MyBioSource (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
96 well plates, by BioLegend (1 mentions)
Elisa reader, by Molecular Devices (1 mentions)
Mouse monoclonal anti myc, by Santa Cruz Biotechnology (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Fusion solo x, by Vilber (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Goat anti rabbit igg, by Promega (1 mentions)
Phospho iκb, by Cell Signaling Technology (1 mentions)
Phospho c src, by Cell Signaling Technology (1 mentions)
Cre recombinase, by Fortrea (1 mentions)
Tmb single component substrate solution, by Solarbio (1 mentions)
9 protocols using hrp conjugated goat anti mouse igg
1
Histological and Western Blot Analysis
2
Western Blot Analysis of Yeast Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extraction of yeast proteins was performed as described elsewhere (Kushnirov, 2000 (link)). Proteins were separated by SDS-PAGE (any kD Criterion TGX stain-free gels, Bio-Rad) and transferred onto Protran 0.2 μm pore nitrocellulose membrane (Perkin Elmar). Blots were probed with the following antibodies diluted in TBS-T buffer containing 5% nonfat dry milk: mouse anti-HA (1:2,000; Sigma H3663), rabbit anti GAPDH (1:1,000; abcam ab9485), HRP conjugated goat anti-mouse IgG (1:10,000; Promega W4021), HRP conjugated goat anti-rabbit IgG (1:10,000; Promega W4011). Blots were developed using SuperSignal West Dura Extended Duration Substrate (Pierce, Life Technologies) and chemiluminescence was detected on a ChemiDoc XRS + imager (Bio-Rad).
3
Decorin and Dermatan Sulfate Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative ELISA for decorin and dermatan sulfate binding by DbpA proteins was performed similarly to that previously described [54] (link). One µg of decorin, dermatan sulfate, chondroitin 6 sulfate, or BSA was coated onto microtiter plate wells. One hundred microliters of increasing concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 µM) of histidine-tagged RevA (negative control) or a DbpA variant, including DbpAB31, DbpA297, DbpAN40-D10/E9, DbpAB356, DbpAVS461, DbpAPBr, or DbpAVS461ΔC11, were then added to the wells. To detect the binding of histidine-tagged proteins, mouse anti-histidine tag (Sigma-Aldrich, St. Louis, MO; 1∶200) and HRP-conjugated goat anti-mouse IgG (Promega, Fitchburg, WI; 1∶1,000) were used as primary and secondary antibodies. The plates were washed three times with PBST (0.05% Tween20 in PBS buffer), and 100 µl of tetramethyl benzidine (TMB) solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were added to each well and incubated for five minutes. The reaction was stopped by adding 100 µl of 0.5% hydro sulfuric acid to each well. Plates were read at 405 nm using a Synergy HT ELISA plate reader (BioTek, Winooski, VT). To determine the dissociation constant (KD), the data were fitted by the following equation using KaleidaGraph software (Version 4.1.1 Abekbecj Software, Reading, PA).
4
Quantitative ELISA for Mouse FH Binding by CspZ Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative ELISA for mouse FH binding by CspZ proteins was performed similarly to that previously described (28 (link)). Basically, 1 µg of BSA (negative control) or mouse FH (MyBiosource, San Diego, CA, USA) was coated onto microtiter plate wells. One hundred microliters of increasing concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 µM) of GST (negative control) or a GST tagged wild-type or mutant CspZ protein, including CspZ or CspZ-Y207A/Y211A were then added to the wells. To detect the binding of GST-tagged proteins, mouse anti-GST tag (Sigma-Aldrich, St. Louis, MO, USA; 1:200) and HRP-conjugated goat anti-mouse IgG (Promega, Madison, WI, USA; 1:1,000×) were used as primary and secondary antibodies. The plates were washed three times with PBST (0.05% Tween 20 in PBS), and 100 µL of tetramethyl benzidine (TMB) solution (ThermoFisher, Waltham, MA, USA) were added to each well and incubated for 5 min. The reaction was stopped by adding 100 µL of 0.5% hydro sulfuric acid to each well. Plates were read at 405 nm using a Tecan Sunrise Microplate reader (Tecan, Morrisville, NC, USA).
5
X31 Virus-Specific IgG Antibody Quantification
6
MERS-CoV Structural Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected cells were lysed in Triton X-100 lysis buffer (1% Triton X-100, 50 mM Tris–Cl [pH 8.0], 150 mM NaCl, 1 mM EDTA) on ice and cleared by centrifugation at 1,000 × g for 10 min at 4 °C. The concentrated VLPs and transfected cell lysates were mixed with SDS solubilizer. Samples were heated at 95 °C for 10 min, separated in 8% or 15% (wt/vol) polyacrylamide-SDS gels, transferred to PVDF membranes, and probed with polyclonal mouse anti-MERS-CoV S (Sino Biological, 1:1000), polyclonal rabbit anti-MERS-CoV M (GeneTex, 1:1000), polyclonal rabbit anti-MERS-CoV E (GeneTex, 1:1000), or monoclonal mouse anti-myc (Santa Cruz, 1:1000) antibodies. The membranes were then probed with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Promega, 1:1000) or anti-rabbit IgG (Bioss, 1:1000) and incubated with ECL substrate (Thermo Fisher Scientific), and the signals were detected using a Fusion Solo X (Vilber). Band density on western blot membranes was analyzed using Evolution Capt software.
7
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with ice-cold PBS, and proteins were extracted at 4°C with M-PER, an inhibitor cocktail. Equivalent amounts of protein were subjected to SDS-PAGE with 7.5–15% Tris-Glycin gradient gel or 15% Tris-Glycin gel and transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). After blocking with 6% milk/TBS-T, membranes were incubated with primary antibodies to Stat5a, Stat5b, RNA polymerase (Santa Cruz Biotechnology, Inc.), Cre recombinase (Covance), phospho-Stat5, cleaved caspase-3, ERK1/2, phospho-ERK1/2, JNK, phospho-JNK, p38, phospho-p38, phospho-Akt, Akt, phospho-IκB, IκB, phospho–c-Src (Cell Signaling Technology), c-Src (EMD Millipore), and β-actin (Sigma-Aldrich), followed by HRP-conjugated goat anti–mouse IgG and goat anti–rabbit IgG (Promega). Immunoreactive bands were visualized with ECL (GE Healthcare) according to the manufacturer’s instructions. The blots were stripped by incubating for 20 min in stripping buffer (2% SDS, 100 mM 2-mercaptoethanol, and 62.5 mM Tris-HCl, pH 6.7) at 50°C and then were reprobed with other antibodies.
8
SARS-CoV-2 S1 Protein Antibody Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum samples from various groups of mice were analyzed using standard indirect Enzyme-linked immunosorbent assay (ELISA) to assess the IgG and its isotype-specific antibody levels of S1 or N proteins. The purified S1 protein (WT, Beta, Delta, Lambda, and Omicron) was coated into ELISA plates by overnight incubation at 4 ℃ for subsequent binding assay. The next day, each well was blocked with 5% skim milk in PBST (PBS with 0.05% Tween-20) for 2 h at 37 ℃. Then, the serum was performed as a primary antibody and serially diluted with ELISA-blocking buffer was incubated for 1 h at 37 ℃. The S1-specific IgG, IgG1, and IgG2a antibodies were titrated by incubating with the horseradish peroxidase (HRP)‐conjugated Goat Anti-Mouse IgG (Promega; W4021), IgG1 or IgG2a (Jackson ImmunoResearch Laboratories; 115–035-205, 115–035-206) as detection antibodies for 1 h at 37 ℃. Each well was developed with TMB Single-Component Substrate solution (Solarbio; PR1200) in the dark for 10 min. 2 M H2SO4 was used to stop color development. Finally, the absorbance at 450 nm was performed immediately using a multi-functional enzyme standard instrument (Omega, Germany). Similar to the Method described above, indirect ELISA was employed to ascertain the endpoint titers of N-protein-specific IgG, IgG2a, and IgG1.
9
Western Blot Analysis of mBAP31 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates (20 μg) were separated by SDS-PAGE and transferred onto Immobilon-P membrane (Millipore, Billerica, MA). The membrane was then blocked with 5% skim milk in PBST (0.29% Na2HPO4, 0.8% NaCl, 0.02% KCl, and 0.05% Tween-20, pH7.4) for 1 h at room temperature (RT) and then incubated with anti-mBAP31 mAb (diluted to 5 μg/mL) or control mAb for 1 h at RT. After washing three times with PBST, the membrane was incubated with HRP-conjugated goat anti-mouse IgG (1:1500 v/v, Promega) for 1 h at RT. Finally, the membrane was developed using an ECL kit (GE Healthcare) and exposed to Agfa x-ray film. Anti-β-actin mAb was used as internal standard for all samples.
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!