Protein samples were extracted from cells and mitochondria, respectively, using RIPA lysis buffer (Chromotek) supplemented with protease inhibitors. Protein samples were diluted in LDS sample buffer (4×, Invitrogen) supplemented with sample reducing agent (10×, Invitrogen). Samples were heated at 95̊C for 5 min before loading on a 4–12% gradient Bis-Tris gel (Invitrogen). MOPS SDS running buffer (Invitrogen) was used and supplied with antioxidants (Invitrogen). The gel was soaked in running buffer and run at 160 V for 75 min. The gel was transferred to a 0.45 µm nitrocellulose membrane (Cytiva) and protein at 30 V for 150 min, 4̊C. The membrane was blocked for 1 h in 5% skimmed milk (Sigma) in PBS-Tween 20 (0.1%, Sigma). The membrane was probed for anti-GFP (ab183734, abcam), anti-mCherry (1C51, ab125096, abcam), anti-VDAC1 (ab15895, abcam) and anti-α tubulin (DM1α, T6199, Sigma), diluted 1 : 10 000, 1 : 3000, 1 : 1500, 1 : 1500, respectively, in 5% milk (PBS-Tween). Primary antibody signal was detected using goat anti-mouse IgG conjugated to IRDye 680RD (ab216776, abcam) and goat anti-rabbit IgG conjugated to IRDye 800CW (ab216773, abcam) secondary antibodies, diluted 1 : 15 000, imaged with an Odyssey CLx imaging system (LI-CO Biosciences).
Ab216773
Manufactured by Abcam
Sourced in United States, China
Ab216773 is a monoclonal antibody that binds to the human XYZ protein. It is intended for use in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab216776, by Abcam (7 mentions)
Odyssey infrared scanner, by LI COR (5 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Irdye 800cw, by Abcam (4 mentions)
Ab8245, by Abcam (3 mentions)
Ab216772, by Abcam (3 mentions)
Ab8226, by Abcam (3 mentions)
Enhanced bca protein assay kit, by Beyotime (2 mentions)
Ab9485, by Abcam (2 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Sample reducing agent, by Thermo Fisher Scientific (1 mentions)
0.45 m nitrocellulose membrane, by Cytiva (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Skimmed milk, by Merck Group (1 mentions)
Mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
T6199, by Merck Group (1 mentions)
Ab183734, by Abcam (1 mentions)
23 protocols using ab216773
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Protein Expression
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Total protein was extracted from indicated cultured cells with RIPA lysis buffer (Beyotime) added to a protease inhibitor PMSF (Beyotime). The concentrations of extracted proteins were detected using Enhanced BCA Protein Assay Kit (Beyotime). Equal amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the separated proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Beyotime). After blocking using fat free milk, the membranes were incubated with primary antibodies against KIAA1522 (ab122203, 1:500, Abcam), ETS1 (ab220361, 1:1,000, Abcam), SNAI1 (#3879, 1:1,000, Cell Signaling Technology, Boston, USA), or GAPDH (ab8245, 1:10,000, Abcam) overnight at 4°C. After being washed using TBST three times, the membranes were further incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773, 1:10,000, Abcam) or Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776, 1:10,000, Abcam) for 1 h at room temperature and then imaged using the Odyssey infrared scanner (Li-Cor, Lincoln, NE, USA).
3
Western Blot Analysis of WNT3A and β-Catenin
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Total cell extracts from indicated cells were obtained using the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China). Protein concentration was determined by the Bicinchoninic acid (BCA) kit (Beyotime). After being separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the protein was transferred onto nitrocellulose membrane. After being blocked by 5% bovine serum albumin (BSA) for 2 h at room temperature, the membrane was incubated with primary antibodies against WNT3A (ab81614, 1 : 1000, Abcam, Cambridge, MA, USA), β‐catenin (ab32572, 1 : 2500, Abcam), or β‐actin (T0022, 1 : 10 000, Affinity, Changzhou, China) overnight at 4 °C. After three rinses, the membrane was incubated with goat anti‐mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776, 1 : 10 000, Abcam) or goat anti‐rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773, 1 : 10 000, Abcam) for 1 h at room temperature. Finally, the membrane was detected with an Odyssey infrared scanner (Li‐Cor, Lincoln, NE, USA).
4
Western Blot Analysis of LRP6 Protein
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The total protein of each group was extracted using RIPA lysis buffer (Beijing solarbio science & technology co., Ltd, Beijing, China) according to the manufacturer’s instructions. Equal amounts of protein from each group of cells were subjected to SDS-PAGE electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane. After being blocked for 1 h at room temperature in 5% fat-free milk, the membrane was incubated with specific primary antibody at 4 °C overnight. The primary antibody was obtained from Abcam (Shanghai, China): Anti-LRP6 (Abeam, ab134146, 1:500). Then the membrane was incubated with an horse radishperoxidase (HRP)-labeled secondary antibody (Abcam, ab216773, 1:5000) at room temperature for 1 h. After the procedure of wash, the membrane was exposed with ECL chromogenic reagent (Millipore, Bedford, MA, USA), and then was exposed to film to observe the bands.
5
Western Blot Analysis of Osteogenic Markers
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Total protein (50 μg) was separated in a Bis-Tris polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was then incubated in 5% bovine serumal bumin (BSA) containing primary rabbit-anti-human polyclonal antibodies at 4°C overnight. Next, samples were incubated with IRDye®800CW goat-anti-rabbit antibody (1:5000; ab216773, Abcam) at room temperature for 1 hour and visualized via chemiluminescence with an infrared laser scanning system (OdysseyLicor, Lincoln, NE, USA). The following primary rabbit-anti-human antibodies were used: anti-FOXO1 (1:1000; ab39670, Abcam); anti-Runx2 (1:1000; ab23981, Abcam); anti-Sp7/Osterix (1:2000; ab22552, Abcam); anti-ALP (1:2000; ab95462, Abcam); anti-OCN (1:500; ab93876, Abcam); anti-OPN (1:1000; ab8448, Abcam) and anti-GAPDH (1:2500; ab9485, Abcam).
6
Quantifying PlyCB Binding to E. coli
7
Immunofluorescence Visualization of LC3A/B
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The detailed methods were described in previous study [38 (link)]. Sections were deparaffinized, hydrated, subjected to antigen retrieval, and blocked as described above (no peroxidase inactivation was performed). Sections were incubated with the primary antibody overnight at 4 °C: rabbit anti-LC3A/B (4108 s, Cell Signaling Technology; 1:200 dilution). Sections were washed as described above, then incubated at room temperature for 1 h in the dark with AlexaFluor 647-linked goat anti-rabbit secondary antibody (ab216773, Abcam). Sections were washed with PBS, sealed with 4′,6-diamidino-2-phenylindole (8961, Thermo Fisher Scientific), stored at 4 °C in the dark, and photographed by fluorescence microscopy (Evos FL Auto2, Thermo Fisher Scientific).
8
Western Blot Analysis of KLF5 and GAPDH
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Total proteins were isolated from cells with RIPA lysate buffer (P0013C, Beyotime, Jiangsu, China). BCA Protein Assay Kit (23227, Thermo Fisher Scientific) was used to determine the concentrations of the protein samples, and the samples were quantified according to different concentrations. Thereafter, the proteins were boiled for 10 min at 95 °C with 10 μL of loading buffer (1610737, Haoran Biological Technology, Shanghai, China) and then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 100V. After electrophoresis, the proteins were transferred onto nitrocellulose membranes, which were sequentially blocked by 5% BSA/TBST for 60 min. Primary rabbit polyclonal antibodies KLF5 (ab137676, 1:2000, Abcam, Cambridge, USA) and GAPDH (ab181602, 1:1000, Abcam) were added onto the membranes for incubation overnight at 4°C, followed by secondary antibody goat anti-rabbit IgG H&L (ab216773, 1:1500, Abcam) at room temperature for 1 h. The membranes were washed with TBST buffer for 3 times. An Enhanced Chemiluminescence (ECL) kit (ECL808-25, Biomiga, USA) was used for the visualization of protein bands, and the Image Pro Plus 6.0 (Media Cybernetics, USA) software was applied to analyze the relative protein levels. The experiment was repeated 3 times independently.
9
Protein Expression Analysis by Western Blotting
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Cells were lysed in CelLytic M (Sigma-Aldrich, Dorset, UK) supplemented with protease and phosphatase inhibitors (Roche, Mannheim, Germany). A 20 µg amount of lysate was separated on 3–8% gradient NuPAGE Tris-Acetate gels (Invitrogen, Waltham, MA, USA) and transferred to nitrocellulose membranes (LI-COR, Lincoln, NE, USA). Revert 700 Total Protein Stain (LI-COR) was used to assess the transfer and for immunoblot normalisation. The membranes were blocked with Intercept (TBS) Blocking Buffer (LI-COR) before probing with the specified antibodies diluted in Intercept T20 (TBS) antibody Diluent (LI-COR). Primary antibodies were used at the following titres: 1:1500 for rabbit monoclonal anti-sacsin (abcam, Cambridge, UK; #ab181190), 1:5000 for mouse monoclonal anti-GAPDH (Invitrogen, #MA-15738), and 1:1000 for mouse monoclonal anti-Hsp70 (abcam; #ab2787) and rabbit polyclonal anti-pan-AKT (abcam; #ab8805). The secondary antibodies used were goat anti-mouse IRDye 680RD (abcam; #ab216776) and goat anti-rabbit IRDye 800CW (abcam; #ab216773). Images were acquired using an Odyssey CLx Imaging System (LI-COR), and analysis was performed using the Empiria Software (version 2.1.0, LI-COR).
10
Western Blot Analysis of Neuronal Proteins
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For western blot analysis, samples were first separated by SDS-PAGE and then electrophoretically transferred onto membranes. After transfer, the membranes were then treated with blocking buffer and labeled using an iBind Flex (ThermoFisher Scientific). GluA1 (Abcam – ab1504, rabbit, 1:2,000, Cambridge, United Kingdom), GluN1 (Synaptic Systems – 114-003, rabbit, 1:1,000), PSD-95 (Abcam – ab18258, rabbit, 1:2,000), VGLUT1 (Abcam – ab77822, rabbit, 1:1,000), Lamp1 (Proteintech – 21997-1-AP, rabbit, 1:2,000, Rosemont, IL, United States), and golgin (Abcam – 84380, rabbit, 1:2,000) were each individually probed. A goat-anti rabbit secondary antibody conjugated with HRP was used for all chemiluminescent western blots (Abcam – ab672, 1:50,000), and a goat-anti rabbit secondary antibody conjugated with IRDye 800CW was used for all fluorescent western blots (Abcam – ab216773, 1:50,000. The bands were visualized either by immunofluorescence with a LI-COR Odyssey (Lincoln, NE, United States) or with chemiluminescence with a Konica Minolta – SRX101A (Tokyo, Japan). All antibodies were diluted from 1 mg/ml stock.
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