Salivary glands (20 glands per well) were separated by SDS-PAGE on 12% polyacrylamide gels under denaturing conditions using the Mini-ATTO apparatus (Biogen). Proteins were either stained by Coomassie brilliant Blue (Bio-Rad) or transferred to PVDF membranes that were cut into strips (area corresponding to one well of the 12 well-comb was cut into 4 strips) and blocked overnight at 4°C with 5% milk in Tris-Tw buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween® 20; pH 7.4) at 4°C. After washing in Tris-Tw, membranes were incubated for 1 h with diluted sera (mice: 1:100; rabbit: 1:50). The strips were further washed and incubated with peroxidase conjugated goat anti-mouse IgG (1:500, AbD Serotec) or goat anti-rabbit IgG (1:2.500, AbD Serotec). Immunogenic protein bands were visualized by substrate solution with diaminobenzidine and reaction was stopped with distilled water.
Goat anti mouse igg
Manufactured by Bio-Rad
Sourced in United States
Goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) molecules. It is commonly used in immunoassays and other immunological techniques to detect and quantify mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Bio-Rad (40 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Bio-Rad (9 mentions)
Re blot plus strong solution, by Merck Group (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Amersham ecltm prime western blotting detection reagent, by GE Healthcare (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Amersham imager 680 system, by GE Healthcare (3 mentions)
Enhanced chemiluminescence western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Lsm 780, by Zeiss (2 mentions)
Phospho eif2α, by Abcam (2 mentions)
Phospho ire1α, by Thermo Fisher Scientific (2 mentions)
Phospho irf3, by Abcam (2 mentions)
Sting, by Abcam (2 mentions)
Tollip, by Abcam (2 mentions)
Sting agonists, by InvivoGen (2 mentions)
Mavs pathway agonist, by InvivoGen (2 mentions)
Irak1, by Thermo Fisher Scientific (2 mentions)
85 protocols using goat anti mouse igg
1
SDS-PAGE and Western Blot Analysis of Salivary Gland Proteins
2
Anti-Saliva Antibody Response Quantification
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Specific anti-P. perniciosus saliva antibody response was measured by indirect ELISA, according to Rohoušová et al. with minor modifications [21 (link)]. Sera were diluted (1:50) in 2% skimmed milk and incubated at 37°C during 3 h. For mouse IgG subtype studies (IgG and IgG2a) sera were diluted 1:200. Peroxidase-conjugated antibodies were incubated for 1 h at 37°C (goat anti-mouse IgG 1:500, goat anti-mouse IgG1 1:10.000, goat anti-mouse IgG2a 1:1.000, goat anti-rabbit IgG 1:2.500; AbD Serotec and goat anti-rabbit IgM 1:20.000; Bethyl Laboratories). Plates were developed with orthophenylendiamine in McIlwein phosphate-citrate buffer (pH 5.5) in the presence of H2O2. The reaction was stopped with 10% H2SO4 and absorbance was measured at 492 nm using an ELISA reader (ELx800, BioTek, Winooski, VT). Each serum was tested in duplicate. The control wells were coated with SGH, but no serum was added. IgG antibody levels were reported as adjusted optical density (aOD), calculated for each serum as a mean OD value of the duplicated wells minus the OD value of the control wells. ELISA experiments were repeated at least twice.
3
Immunofluorescence Staining of Kidney Tissue
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Kidneys were embedded in OCT compound and frozen at −70 °C. Samples were cut into 5-μm-thick sections, fixed in 4% paraformaldehyde, and washed 3 times in cold PBS. Nonspecific binding was blocked with 1% normal goat serum in PBS with Tween 20 for 30 min. Sections were incubated with goat anti-mouse IgG (1:100; Serotec, Oxford, UK) and rabbit anti-mouse complement 3 (C3) (1:100; Abcam) at 4 °C overnight, washed 3 times with PBS, incubated with Alexa Fluor 568–labeled donkey anti-goat IgG and Alexa Fluor 488-labelled donkey anti-rabbit IgG (each 1:100; Invitrogen) at room temperature for 1 h, and washed 3 times with PBS. Sections were mounted in a mounting solution (Vector Laboratories, Burlingame, CA, USA) and examined under a laser scanning confocal microscope (LSM 780; Carl Zeiss, Oberkochen, Germany). Two pathologists independently assigned semi-quantitative scores indicating the intensity and distribution of immunofluorescence staining for IgG and C3, on a scale ranging 0–3, in which 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The average score obtained by the 2 pathologists was determined.
4
Western Blot Analysis of Paxillin and PARP-1
5
Quantifying Antigen-Specific IgG Response
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Two days before each vaccine and 15 days after the final immunization, the serum IgG (IgG1, IgG2a) of each sample was measured using indirect ELISAs. The purified rBLS-Usp45 was employed to coat the wells of a polycarbonate plate with 100 μL of a 5 μg/mL solution in carbonate buffer (pH 9.6). To prevent non-specific binding, the plates were inhibited with 5% skim milk in PBS containing 0.5% Tween 20 for an additional overnight incubation at 4◦C after being rinsed with PBS containing 0.05% (w/v) Tween 20. Plates were incubated with 100 μL of diluted serum samples diluted 1:100 in preventing buffer for 2 h at room temperature while being rocked after the blocking buffer was removed. Goat Anti-Mouse IgG (Bio-Rad Cat no: 170-6516) labeled with HRP was incorporated into the wells at a dilution of 1:2000 in PBS-Tween and cultured at room temperature for one hour after three further PBS-Tween washes were conducted between incubations. Following a final washing procedure, the administration of 50 μL/well of the enzyme–substrate TMB for 30 min at 37 C was used to measure specific reactivity. When 2 M H2SO4 was added to each well, the reaction was halted. After 10 min, the ideal density (OD 492) at 492 nm was determined. Every test was run in triplicate.
6
ER Stress and Immune Signaling Pathways
7
SARS-CoV-2 S Protein Antibody ELISA
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Binding antibodies were assessed by ELISA. Briefly, 96-well plates (Corning, Cat.9018) were coated at 4 °C overnight with 1.0 mg/mL S protein in phosphate-buffered saline (PBS). Plates were washed once with PBST (0.05% Tween 20 in PBS) and blocked with 5% milk-PBS for 2 h at 37 °C. Five-fold serial dilutions of heat-inactivated serum were made in 2% milk-PBS for 100 μL/well and plates were incubated at 37 °C for 1 h. This was followed by 1 h incubation at 37 °C with a dilution of horseradish peroxidase (HRP) conjugated Goat anti-mouse IgG (1:10000) (Bio-Rad, Cat.1706516), Goat anti-hamster IgG (1:15000) (Invitrogen, Cat.PA1-28823), or Goat anti-monkey IgG (1:5000) (BETHYL, Cat.A140-102P). Plates were then washed six times, 100 μL per well of TMB substrate system solution (SeraCare, Cat.5120–0038) was added for 15 min in the dark and stopped by adding 50 μL 2 M sulfuric acid. The absorbance was measured as OD450-OD620 using a microplate reader (Molecular Devices, SpectraMax iD3).
The endpoint titers were defined as the highest reciprocal dilution of serum to give an absorbance >2.1-fold of the background values that defined as 0.05. Antibody titer below the limit of detection was determined as half the limit of detection.
The endpoint titers were defined as the highest reciprocal dilution of serum to give an absorbance >2.1-fold of the background values that defined as 0.05. Antibody titer below the limit of detection was determined as half the limit of detection.
8
Apigenin Modulates JAK2/STAT3 Signaling
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Antibodies against phospho-STAT3 (Tyr705), STAT3, phospho-JAK2 (Y1007/1008), JAK2, phospho-Src (Tyr 416), Src, Keratin and Fibronectin were obtained from Cell Signaling Biotechnology (Beverly, MA, USA). Antibodies against Twist1 and β-actin, and Twist1 small interfering RNA (Twist1-siRNA) were purchased from Santa Cruz Company. Goat anti-rabbit IgG, goat anti-mouse IgG and protein marker were supplied by Bio-Rad (Hercules, CA, USA). Antibody against N-cadherin was purchased from BD Biosciences (San Jose, CA, USA). Other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Apigenin was obtained from Shanghai R&D Center for Standardization of Chinese Medicines (Shanghai, China, purity >99% as determined by HPLC). The stock solution of 100 mM apigenin was prepared in dimethyl sulfoxide (DMSO).
9
Western Blot Analysis of YAP Expression
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Western blots were carried out as previously described48 (link)49 (link). Briefly, 50–100 μg of protein from whole cell extracts from both tumor and non-neoplastic liver were re-suspended in 4X tris-glycine sample buffer, boiled at 95 °C for 5–10 min, and fractionated on a 4–20% tris-glycine gel (Invitrogen, Carlsbad, CA) for 90 min at 125–130 V. Proteins were transferred to PVDF membranes and then blocked with a solution of either 5% BSA or 3% BSA and 5% milk in tris-buffered saline with Tween 20 (TBST) before immunodetection with the following antibodies: anti-YAP (Cell Signaling #4912, Danvers, MA), and anti-GAPDH (Millipore #MAB374, Billerica, MA). Overnight primary antibody incubation was followed by washes (60 min at room temperature) in TBST before incubation in secondary antibody for 1 hour (horseradish peroxidase-conjugated goat anti-rabbit IgG, Cell Signaling; or goat anti-mouse IgG, Bio-Rad, Hercules, CA). After washing with TBST, proteins were visualized using the ECL detection system (NEN, Boston, MA). Coomassie gels were used to ensure equal protein loading. Band densities were analyzed using AlphaEase FC software version 4.1.0 (Alpha Innotech) to determine relative protein expression, and were then normalized to GAPDH band densities.
10
Western Blotting of Cultured Cells
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Cultured cells were lysed in RIPA buffer (150 mM NaCl, 10 mM Tris–HCl pH 7.4, 0.1% SDS, 1% DOC, 5 mM EDTA, 1% Triton X-100 and 1× EDTA-free Proteinase Inhibitor Cocktail (PIC) from Roche 11873580001) for 10 min on ice, homogenized and centrifuged at 14000g/4ºC for 10 min. For western blotting, the following commercial primary antibodies were used: anti-ß-ACTIN (Santa Cruz, sc-47778), anti-CELF1 (Santa Cruz, sc-20003), anti-PDIA6 (ThermoFisher PA3-008 or Proteintech 18233–1-AP), anti-Vinculin (Sigma V9131), anti-HA tag (Abcam ab9110), anti-GFP (Invitrogen A6455), anti-Ras (BD Biosciences 610001), anti-α-Tubulin (Sigma T9026). Anti-Stubarista and anti-CSDE1 were produced in house (24 (link),28 (link)). For chemiluminescence detection, we used Goat Anti-Rabbit IgG and Goat Anti-Mouse IgG (Biorad, 1706515 and 1721011), and for infrared (IR) detection, IRDye 800CW and IRDye 680RD (LI-COR, 92632213 and 92668072). The Gels tool from ImageJ was used for quantification.
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