The presence of serum IgG antibodies that block binding of NoV VLPs to the HBGA carbohydrates were determined in ELISA-based blocking assay according to previously published protocols [23 (link)]. Group-wise pooled mouse sera were examined for capability to block VLP binding on HBGAs present in pig gastric mucin (PGM, type III, Sigma-Aldrich, Saint Louis, MO, USA, Cat. M1778) [23 (link),34 (link)]. Serum two-fold dilutions were pre-incubated with 0.1 µg/mL GI.4 or GII.4-2006a NoV VLPs in sample buffer (1% milk in PBS + 0.05% tween) prior to plating on PGM-coated (2 µg/mL) and blocked (5% milk in PBS) microwell plates (Corning Inc., Corning, NY, USA, Cat. 3690). Following 1 h incubation at +37 °C, bound VLPs were detected with rabbit polyclonal anti-NoV antisera (ICON Genetics, Halle, Germany) followed by anti-rabbit IgG-HRP antibody (Abcam, Cambridge, UK, Cat. ab97051) and OPD substrate. Maximum HBGA binding of VLPs was determined in wells with VLPs lacking the serum. The blocking index (%) was calculated as follows: 100% − [(OD490 of wells with VLP and serum/OD490 of maximum binding wells) × 100%]. A 50% blocking titer (BT50) was determined as the reciprocal of the highest serum dilution able to block at least 50% of VLP-HBGA binding.
Hrp anti rabbit igg antibody
Manufactured by Abcam
Sourced in United Kingdom
The HRP Anti-Rabbit IgG antibody is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to rabbit primary antibodies, enabling signal amplification in immunoassays and immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab170904, by Abcam (3 mentions)
Ab79823, by Abcam (3 mentions)
Ab92544, by Abcam (2 mentions)
Ab260038, by Abcam (2 mentions)
Ab182858, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Pgm type 3, by Merck Group (1 mentions)
Anti ampk, by Abcam (1 mentions)
Anti p mtor, by Abcam (1 mentions)
Anti beclin1, by Abcam (1 mentions)
Anti gadph, by Abcam (1 mentions)
Anti p akt, by Abcam (1 mentions)
Anti p ampk, by Abcam (1 mentions)
Anti atg5, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Anti mtor, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti akt, by Abcam (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
9 protocols using hrp anti rabbit igg antibody
1
Serum IgG Antibody Blocking Assay for NoV VLPs
2
Protein Isolation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adioimmunoprecipitation assay (RIPA) buffer was used to isolate the protein by protein lysis, and the protein dose was estimated with a bicinchoninic acid (BCA) protein assay kit (Beyotime). Then, whole protein was obtained by Sodium dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Samples were intercepted with 3% bovine serum albumin including Tween-20 and 0.05% Tris-buffered saline. Membrane was incubated with anti-p-AKT (1/500, Abcam), anti-AKT (1/500, Abcam), anti-p-mTOR (1/1000, Abcam), anti-mTOR (1/2000, Abcam), Anti-p-AMPK (1/500, Abcam), Anti-AMPK (1/500, Abcam), Anti-LC3Ι, Π (1/500, Abcam), Anti-Beclin-1 (1/500, Abcam), Anti-Atg5 (1/1000, Abcam), and anti-GADPH (1/500, Abcam) with a secondary anti-rabbit IgG-HRP antibody (1/1000, Abcam) for 1.5 hr at the room temperature. In the end, the strips were labeled with DAB (Sigma, Germany). Also, protein presentation was analyzed utilizing Image J software (Ashrafizadeh et al., 2019 ) .
3
MCM2 Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First, 5×105 HeLa and SiHa cells were suspended in lysis buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5 mM MgCl2, 10% glycerol, 1% Triton X-100 and 0.1% sodium dodecyl sulfate (SDS)] containing the protease inhibitor cocktail (Roche Diagnostics) on ice for 30 min. Subsequently, the samples were centrifuged for 15 min at 12,000 × g at 4°C. After the supernatants were collected, protein concentrations were measured using the Dc Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc.). Proteins (20 µg/lane) extracted from the cells were separated using a 10% SDS polyacrylamide gel and subsequently transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at 25°C and then incubated overnight at 4°C with the anti-MCM2 antibody (dilution, 1:1,000; cat. no. ab4461; Abcam) and the anti-GAPDH antibody (dilution, 1:1,000; cat. no. ab128915; Abcam) followed by the HRP Anti-Rabbit IgG antibody (dilution, 1:5,000; cat. no. ab270144; Abcam) for 2 h at 25°C. The blots were developed using the Immobilon Western Chemiluminescent HRP Substrate (cat. no. WBKLS0500; Merck KGaA). Densitometry was performed using ImageJ 1.48 (National Institutes of Health).
4
Nuclear-Cytoplasmic Protein Fractionation and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from cells was extracted using RIPA lysate, and nuclear-cytoplasmic separation was performed with the Minute (TM) Cytoplasmic and Nuclear Fractionation kit (SC-003, Invent). Equal amounts of protein were run on SDS-PAGE and electroporated onto PVDF membrane. Membrane was sequentially blocked in 5% bovine serum albumin for 1 h, incubated with primary antibodies (anti-HIF-1α, 1:2000, ab1; anti-Lamin B1, 1:5000, ab16048; anti-GAPDH, 1:5000, ab8245; anti-BIRC7, 1:2000, ab97350; Abcam) overnight at 4°C and then incubated with corresponding secondary antibodies (HRP Anti-Rabbit IgG antibody, 1:3000; ab288151; HRP Goat Anti-Mouse IgG H&L, 1:3000; ab205719; Abcam) for 2 h at room temperature.
5
Immunofluorescence Microscopy of VEGF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% paraformaldehyde and incubated with anti-VEGF (Cat no. 19003-1-AP; Proteintech, Shanghai, China) overnight at 4°C. Then, HRP Anti-Rabbit IgG antibody (Cat no. ab288151; Abcam, Cambridge, United Kingdom) was used. Nuclei were blocked with DAPI for 10 min. Coverslips were observed by an inverted fluorescence microscope (Axiocam 702 mono, Zeiss, Germany).
6
Western Blotting for PLAU Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was used to detect the expression of the PLAU protein according to previous study [19 (link)]. Total protein from transfected cells was isolated using 200 µL of RIPA lysis buffer (Beyotime, China). Then, 20 µg of protein was added to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking the membranes with 5% nonfat milk in TBST, the membranes were incubated with PLAU antibody (Cat#: ab169754, Abcam, USA) or GAPDH antibody (Cat#: ab9485, Abcam, USA) at 4°C for 12 h. Next, the HRP anti-rabbit IgG antibody (Cat#: ab270144, Abcam, USA) was incubated at 22°C for 3 h. After washing the membranes, SuperEnhanced chemiluminescence detection reagent (Applygen, China) was added to the membranes for 15 min incubation, and the damp blot was covered with plastic wrap. The blot was exposed to an X-ray film.
7
HMGB1 and P-gp Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, the sections were treated with Triton X-100 and blocked with goat serum. Then, the primary antibody (HMGB1, ab79823, Abcam, UK, 1:400; P-gp, ab170904, Abcam, UK, 1:500) was used to incubate the cells, followed by a secondary antibody (HRP Anti-Rabbit IgG antibody, Abcam, UK, 1:600) incubation. After DAPI staining, the localization was viewed under a confocal laser scanning microscope (Olympus, Japan).
8
Protein Expression Analysis via Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polyvinylidene difluoride (PVDF) membranes were used to transfer the whole protein separated with SDS-PAGE. The primary antibody was added and incubated at 4 °C for overnight and the secondary antibody was incubated at 37 °C for an hour after non-fat milk powder blocked the PVDF membrane for an hour at RT. All antibodies we used were shown as below: HMGB1 (ab79823, Abcam, UK, 1:5000), Caspase-3 (ab184787, Abcam, UK, 1:2000), Bax (ab32503, Abcam, UK, 1:1000), Bcl-2 (ab182858, Abcam, UK, 1:2000), LRP (ab92544, Abcam, UK, 1:5000), MRP (ab260038, Abcam, UK, 1:1000), P-gp (ab170904, Abcam, UK, 1:1000), and the secondary antibodies (HRP Anti-Rabbit IgG antibody, Abcam, UK, 1:8000). Chemiluminescence substrate (Boster, China) was used to determine the gray value.
9
Immunohistochemical Analysis of Drug Resistance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following heat-activated antigen extraction, sections were stained immunohistochemically according to the preceding description [22 (link)]. The following primary antibodies were used: HMGB1 (ab79823, Abcam, UK, 1:400), Bax (ab32503, Abcam, UK, 1:250), Bcl-2 (ab182858, Abcam, UK, 1:400), lung resistance protein (LRP, ab92544, Abcam, UK, 1:100), multidrug resistance-related protein (MRP, ab260038, Abcam, UK, 1:500), P-glycoprotein (P-gp, ab170904, Abcam, UK, 1:500), and the secondary antibody, HRP Anti-Rabbit IgG antibody (Abcam, UK, 1:600), was utilized. Images were taken with an Olympus BH2 microscope (Olympus Corporation, Japan).
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!