Murine sera against each plant virus were obtained through immunization of mice (see above) and tested for potential cross-reactivity against the other plant viruses. For this, 6 μg of CPMV, CPSMV, or TRSV was separated on SDS-PAGE and transferred onto the nitrocellulose membrane followed by western blot. All wash steps in between incubations were done with PBST (PBS with 0.05% (v/v) Tween-20), membranes were washed three times at room temperature, and all incubation steps were done for 1 h at room temperature. First, the membrane was blocked in 5% (w/v) milk powder in PBS, washed, and incubated in αCPMV, αCPSMV, or αTRSV mouse sera, respectively, 1:1000 in PBS. After washing, the membrane was incubated with a HRP-labeled goat anti-mouse IgG (Thermo Fisher Scientific), 1:5000 in PBS. After a final wash, the DAB substrate [3,3′-diaminobenzidine; DAB Susbtrate Kit, Peroxide (HRP), Vector Laboratories] was added to the membrane and incubated for 1–5 mins or until bands were visible. The reaction was stopped with water and the membranes immediately imaged under white light with a FluorChem R imaging system (Protein Simple).
Hrp labeled goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
HRP-labeled goat anti-mouse IgG is a secondary antibody reagent that binds to mouse immunoglobulin G (IgG) antibodies. The antibody is conjugated with horseradish peroxidase (HRP), which can be used for detection and quantification applications in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Hrp labeled goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Bca assay, by Beyotime (1 mentions)
Ab109012, by Abcam (1 mentions)
Ab108327, by Abcam (1 mentions)
Electrochemiluminescence kit, by Beyotime (1 mentions)
Ab48394, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Electrochemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Anti β actin, by Affinity Biosciences (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti ps6 ser240 244, by Cell Signaling Technology (1 mentions)
Anti ps6, by Cell Signaling Technology (1 mentions)
8 protocols using hrp labeled goat anti mouse igg
1
Evaluating Plant Virus Cross-Reactivity
2
Extraction and Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins were extracted from the cells at 4°C for 30 min. After centrifugation at 10,000 g for 10 min, the total proteins were collected. The protein concentration was determined bicinchoninic acid assay (BCA) assay (Beyotime, Beijing, China). Thereafter, the proteins were run on SDS-PAGE for 1–2 h and transferred to a nitrocellulose membrane. The membranes were blocked in 5% defat milk at room temperature for 2 h. After that, the membranes were incubated with the following primary antibodies at 4°C overnight: mouse monoclonal anti-GAPDH (TA-08, 1/2000, ZSbio); rabbit polyclonal anti-LC3 (ab48394, 1/1000, Abcam); rabbit polyclonal anti-p62 (ab109012, 1/10,000, Abcam); and rabbit polyclonal anti-ATG5 (ab108327, 1/5000, Abcam). Thereafter, the membranes were incubated with the following secondary antibodies at 4°C for 2 h: HRP-labeled goat anti-rabbit IgG (cat. no. 65–6120; Thermo Fisher Scientific, Inc.) or HRP-labeled goat anti-mouse IgG (cat. no. 31,430; Thermo Fisher Scientific, Inc.). The bands were visualized using an electrochemiluminescence kit (Beyotime, Beijing, China). The quantification of protein expression was conducted using the Quantity One software (v4.62; Bio-Rad Laboratories, Inc.) with β-actin as the loading control as previous described.16 (link)
3
Western Blot Analysis of GALNT8 and β-actin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary antibodies are GALNT8 (Abcam, #ab121374) and β-actin (Cell signaling technology, #3700). The secondary antibodies are HRP-labeled goat anti-rabbit IgG (Thermo Fisher, #31460) and HRP-labeled goat anti-mouse IgG (Thermo Fisher, #31430). Oxaliplatin was purchased from MCE (#HY-17371).
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following treatment, total protein was extracted from cells using the TriplePrep kit (cat. no. 28-9425-44; ReadyPrep; Cytiva). Total protein was quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). A total of 25 µg/lane protein was separated via SDS-PAGE (10% gel) and transferred onto nitrocellulose membranes, as previously described (24 (link)). The membranes were blocked in 5% skimmed milk at room temperature for 2 h and incubated with the following primary antibodies overnight at 4°C: Rabbit polyclonal anti-Bcl-2 19 kDa interacting protein (BNIP3; 1:1,000; cat. no. ab109362; Abcam), mouse polyclonal anti-apoptosis-inducing factor (AIF; 1:1,000; cat. no. BF0591; Affinity Biosciences) and mouse monoclonal anti-β-actin (1:2,000; cat. no. TA890010; OriGene Technologies, Inc.). The membranes were washed three times with 1X phosphate-buffered saline with 0.05% Tween (PBST) and incubated with secondary antibodies (HRP-labeled goat anti-rabbit IgG; cat. no. 65-6120; Thermo Fisher Scientific, Inc.; HRP-labeled goat anti-mouse IgG; cat. no. 31430; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. Protein bands were visualized using an electrochemiluminescence kit (Thermo Fisher Scientific, Inc.). Protein expression was quantified using the Quantity One software (v4.62; Bio-Rad Laboratories, Inc.) with β-actin as the loading control.
5
Anti-rhMOG IgG ELISA Protocol
6
Western Blot Analysis of Phosphorylated Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Collected cells were washed twice with PBS and then lysed for 30 min on ice in RIPA solution containing protease inhibitor cocktails (Roche, Basel, Switzerland) and phosphatase inhibitors (Sigma, St. Louis, Missouri, USA), as described previously [43 (link)]. Equivalent protein concentrations were subjected to 8%–12% SDS-PAGE (Bio-Rad, Hercules, CA, USA). anti-p-S6 (Ser240/244; catalog no. #2215), anti-p-S6 (Ser235/236; catalog no. #5316), anti-p-Akt (Ser473; catalog no. #4060), anti-p-Akt (Thr308; catalog no. #13842), anti-GAPDH (catalog no. #5174) and anti-β-actin (catalog no. #58169) were purchased from Cell Signaling Technology (Danfoss, Boston, Ma, USA). HRP-labeled goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) and HRP-labeled goat anti-mouse IgG were used.
7
Western Blot Analysis of p53 in GSIV-Infected GSM Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples of GSM cells with GSIV infection (MOI = 0.5) for different time points were prepared as above. After separated by SDS-PAGE, transferred to PVDF membranes and blocked, the membranes were then respectively incubated with an anti-p53 mouse antibody (CST, Danvers, MA, USA) at a dilution of 1:1000, and anti-β-actin antibody (CST, Danvers, MA, USA) at a dilution of 1:1000 at 4 °C overnight. After washed and incubated with HRP-labeled goat-anti-mouse IgG (Invitrogen, Carlsbad, CA, USA), the membranes were detected as above. β-actin was treated as a reference.
8
Quantification of SARS-CoV-2 Antibody Titers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The level of antibody specific to SARS-CoV-2 was quantitated using virion capture ELISA, as previously described [36 (link)]. The plates were pre-coated with rabbit SARS-CoV-2 polyclonal antibody (1:4000, Sino Biological, Beijing, China) overnight at 4 °C, washed, and blocked with 1% BSA in PBST. UV-inactivated SARS-CoV-2 (10⁴ PFU/well) of WT or Delta strain diluted in MEM medium was added into each well and incubated for 2 h. After incubation, diluted samples of immunized mice were applied and incubated. The levels of anti-SARS-CoV-2 antibody were detected using HRP-labeled goat anti-mouse IgG (1:3000, Invitrogen) or IgA antibody (1:4000, Southern Biotech, Birmingham, AL, USA), respectively. The plates were washed and TMB substrate was added for signal development. The reactions were terminated using 1 N HCl, and the absorbances were recorded at 450 nm.
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!