The associations of SARS-CoV-2 nsp13 with PRKACA and CREB1 in lung sections from infected mice and COVID-19 patients were investigated with in situ Proximity Ligation Assay Reagent (Sigma-Aldrich, DUO92008). In brief, after sections were sliced, the sections on glass slides were fixed, permeabilized, and blocked as described above. Then, the sections were incubated with rabbit anti-nsp13 (ABclonal, A20311, 1:50 dilution), mouse anti-PRKACA (BD Biosciences, 610980, 1:100 dilution), or mouse anti-CREB (Abcam, ab178322, 1:100 dilution) primary antibodies. The anti-nsp13 antibody alone was employed as a negative control. After washing three times with PBS, the sections were incubated with affinity-purified donkey anti-rabbit or anti-mouse IgG (Sigma-Aldrich, DUO92002, DUO92004) secondary antibodies. The red fluorescent puncta were generated via a DNA amplification-based reporter system, and nuclei were stained with DAPI (blue). Images were acquired using a laser scanning confocal microscope (Zeiss LSM 800 Meta, built-in software ZEN2.3) with a 63× oil immersion objective lens.
Ab178322
Manufactured by Abcam
Sourced in United States
Ab178322 is a lab equipment product. It is a recombinant human protein.
Automatically generated - may contain errors
Lab products found in correlation
A20311, by BD (2 mentions)
Lsm 800 meta, by Zeiss (2 mentions)
Ab11267, by Abcam (1 mentions)
Ab33922, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions)
Ab108319, by Abcam (1 mentions)
Ab32096, by Abcam (1 mentions)
Ab152, by Merck Group (1 mentions)
Peroxidase conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
4 protocols using ab178322
1
SARS-CoV-2 nsp13 Interactome Mapping
2
Western Blot Analysis of Neuronal Proteins
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Total protein was extracted in cell lysate on ice and the concentration of proteins was tested using the bicinchoninic acid assay kit (Thermo Scientific Pierce, Rockford, IL, USA). Next, the proteins were separated by SDS‐PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were washed with Tris‐buffered saline‐tween (TBST) three times (15 min/time) and blocked with 5% skim milk for 2 h. Afterward, the membranes were cultured with the primary antibodies at 4℃ overnight: brain‐derived neurotrophic factor (BDNF) (ab108319, 1:1000, Abcam Inc., Cambridge, MA, USA), cAMP‐response element‐binding protein1 (CREB1) (ab178322, 1:500, Abcam), GFAP (ab33922, 1:500, Abcam) and MAP2 (ab11267, 1:1000, Abcam). Following washing with TBST (3 times/15 min), the membranes were cultured with the secondary antibody IgG (1:2000, ab205718, Abcam) for 2 h and then washed with TBST (three times/15 min) before chemiluminescence developing and visualization. The image of protein blotting was analyzed by Image J2x v2.1.4.7 software (Rawak Software, Inc. Dresden, Germany).
3
Fluorescence Imaging of SARS-CoV-2 Proteins
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A549 and Caco-2 cells were transfected or infected as described above. Cells on glass coverslips were fixed with paraformaldehyde (PFA; 4%) for 10 min and were then permeabilized with 0.2% Triton X-100 in PBS for 15 min. After blocking with 0.2% goat serum in PBS for 30 min, the cells were incubated with rabbit anti-nsp13 (ABclonal, A20311, 1:50 dilution), mouse anti-PRKACA (BD Biosciences, 610980, 1:100 dilution), or mouse anti-CREB (Abcam, ab178322, 1:100 dilution) antibodies overnight at 4°C. The cells were washed three times with PBS and then incubated with FITC- or TRITC-conjugated goat anti-rabbit or anti-mouse IgG. The cells were then washed, stained with DAPI, and imaged using a laser scanning confocal microscope (Zeiss LSM 800 Meta, built-in software ZEN2.3) with a 63× oil immersion objective lens. The signal intensity was analyzed by Fiji (ImageJ) software (30 (link)).
4
Protein Extraction and Western Blot Analysis
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Fresh tissue was rapidly isolated and stored in a −80 °C freezer for subsequent processing. The VMH was separated from the section under a stereo microscope (L-Z2000, Leica). Tissues were homogenized and lysed in RIPA lysis buffer (89900, ThermoFisher) which contains proteinase inhibitors (04693132001, Roche) and phosphatase inhibitors (4906845001, Roche). The lysates were then washed and boiled in SDS loading buffer. Equal amounts of protein lysates were resolved on SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membrane. The membranes were blocked in 5% bovine serum albumin and incubated with primary antibodies (1: 5000 dilution): rabbit anti-pCREB (phospho S133) (ab32096, Abcam), mouse anti-CREB (ab178322, Abcam), rabbit anti-TH (AB152, Millipore), rabbit anti-Ucp1 (U6382, Sigma-Aldrich) and rabbit anti-GAPDH (2118, Cell Signalling Technology) overnight at 4 °C. After three washes, the membranes were incubated with peroxidase-conjugated anti-rabbit secondary antibody (7074, Cell Signalling Technology) or peroxidase-conjugated anti-mouse secondary antibody (31430, Invitrogen) for 1 h and visualized with enhanced chemiluminescence substrate (34580, Thermo Scientific). The Bio-Rad ChemiDoc system was used to visualize the blots.
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