Protein fractions were isolated using the active motif kit (Active Motif, Carlsbad, CA, USA). Total protein lysates were quantified by Bradford assay. Proteins were separated by 10% SDS-PAGE, transferred onto a nitrocellulose membrane, and blocked overnight in blocking solution (1× TBS, pH 7.6, 0.1% Tween-20, and 5% w/v nonfat dry milk). The membrane was then incubated with antibodies specific for VEGF-A (Ab105846, 1:500; Abcam; Cambridge, MA, USA). As a loading control, the membrane was probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000, NB300-221; NOVUS Biological; Littleton, CO, USA). The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody (1:1,000) in blocking solution for 1 hour at room temperature. Horseradish peroxidase activity was detected by incubating the membrane in chemiluminescence solution (Bio-Rad, Hercules, CA, USA). The exposure time was adjusted to keep the integrated optical densities within a linear and nonsaturated range. Densitometric analysis was carried out using a UVP Bioimaging system (UVP, Minneapolis, MN, USA).
Nb300 221
Manufactured by Novus Biologicals
Sourced in United States
The NB300-221 is a laboratory instrument designed for conducting western blot analysis. It provides a regulated and consistent environment for protein transfer and membrane blocking processes.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Chemiluminescence solution, by Bio-Rad (2 mentions)
Ck 66633, by Bio-Techne (2 mentions)
Stealth select rnai sirna, by Thermo Fisher Scientific (2 mentions)
Epr2536 2, by Novus Biologicals (2 mentions)
Pluronic f 127, by Merck Group (2 mentions)
β lactoglobulin, by Merck Group (2 mentions)
A5441, by Merck Group (2 mentions)
Gapdh, by Novus Biologicals (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Abt128, by Merck Group (2 mentions)
Sc 47734, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Uvp bioimaging system, by Analytik Jena (1 mentions)
Ab992, by Abcam (1 mentions)
S8820, by Merck Group (1 mentions)
24 protocols using nb300 221
1
Western Blot Analysis of VEGF-A
2
GFP-Tagged Protein Complex Isolation
3
Arp2/3 Complex Inhibitor CK-66633 Protocol
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The Arp2/3 complex inhibitor CK-66633 (link) was purchased from Tocris Bioscience and dissolved in DMSO. BSA, β-Lactoglobulin and Pluronic F127 were purchased from Sigma and dissolved in 1x PBS. pLL-PEG was from Surface SolutionS and diluted in 10~mM HEPES. GFP-FAK was provided by Ekaterina Papusheva/ Carl-Philipp Heisenberg. Speckle GFP-Vinculin was a gift from Alan Rick Horwitz. MRLC-GFP was a gift from Rex Chisholm. siRNAs were Stealth Select RNAi siRNAs from Invitrogen with the following sequences: TLN1 siRNA (RSS320275): 5'-GGGCAUAUCCAUGUCUUCGAGCAAA-3'; TLN2 siRNA (RSS321623): 5'-GAGAGGAGCCGAGAAGCGAAUAUUU-3'. Knockdown efficiency was quantified by Western blotting with antibodies for TLN1 (monoclonal, clone 8d4, Sigma #T3287, 1:2000), TLN2 (GeneTex, #EPR2536(2), 1:1000) and GAPDH (clone 1D4, NovusBiologicals #NB300-221, 1:20000). Representative images of immunoblots are based on at least 2 experiments.
4
Arp2/3 Complex Inhibitor CK-66633 Protocol
5
Western Blotting of Cell Signaling Proteins
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Proteins were extracted from cells in RIPA lysis buffer with protease inhibitors (S8820, Sigma, Saint-Louis, MO, USA). Antibodies against CDC20 (1:1000; sc-13162, Santa Cruz, Dallas, TX, USA), hnRNP-U (1:1000; sc-32315), pH3 (1:1000; 06-570; EMD Millipore, Burlington, MA, USA), MPM-2 (1:1000; 05-368; EMD Millipore), cyclin B1 (1:1000; sc-245), cyclin A (1:1000; sc-271682), cyclin E (1:1000; sc-247), PARP (1:1000; 9532; CST), GAPDH (1:1000; NB300-221, Novus Biologicals, Centennial, CO, USA), HSP90 (1:1000; 4877, CST, Beverly, MA, USA), H3 (1:1000; 4499; CST), Myc (1:1000; sc-40), Ubiquitin (1:1000; 43124, CST), CTCF (1:1000; 3418; CST), RAD21 (1:1000; ab992; Abcam, Boston, MA, USA), V5 (1:2000; 80076; CST), Flag (1:1000; 14793; CST), and HA (1:1000; 3724; CST) were used in this study. Antibody against actin (1:5000; A5441, Sigma) was used as a loading control.
6
Western Blot Analysis of Sox18 Protein
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Total protein lysates were isolated and quantified by Bradford assay. The lysates were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (BioRad, Hercules, CA). The membrane was incubated in blocking solution (1x TBS, pH 7.6, 0.1% Tween-20, and 5% w/v of nonfat dry milk) and then incubated with a primary antibody to detect Sox18 (Abcam ab23342). The membrane was probed for GAPDH (NOVUS Biological, NB300-221) to normalize the protein loading. The membrane was then incubated with HRP-conjugated secondary antibody (1 : 1000) in blocking solution for 1 hour at room temperature. HRP activity was detected by incubating the membrane in chemiluminescence solution (Bio-Rad, Hercules, CA). The exposure time was adjusted to keep the integrated optical densities within a linear and nonsaturated range. Densitometric analysis was done using a UVP Bioimaging system (UVP, Minneapolis, MN).
7
Quantification of STAT5 and GAPDH Proteins
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Total protein was extracted in RIPA buffer (50 mM Tris-HCl (PH7.5), 150 mM NaCl, 10% Glycerol, 50 mM NaF, 0.5 mM EDTA and 1% NP-40) and 20 μg of proteins was loaded on 7.5% Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad, #4561023EDU) for STAT5 and in NuPAGE® Novex 4–12% Bis-Tris Gel (Invitrogen, NP0321BOX) for GAPDH. Primary antibodies were incubated overnight at 4°C (anti-STAT5A (Santa Cruz Biotechnology, sc-1081X, 1:1000) and GAPDH (Novus Biologicals, NB300-221, 1:10000)).
8
Tissue Homogenization and Western Blot Analysis
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Protein samples were prepared by homogenizing snap frozen tissue in 1× RIPA (ThermoFisher) buffer containing protease inhibitors (Roche). Protein concentrations were determined using the Bradford assay (BioRad protein assay reagent). Aliquots of total protein (10 μg for aorta and liver; 50 μg for all other tissues) resolved by SDS–PAGE and electro-transferred to nitrocellulose membranes. Blots were blocked with 5% nonfat dry milk in TBST for 1 h at room temperature and then incubated overnight with primary antibody. After rinsing, the membranes were incubated with HRP-conjugated secondary antibodies (BioRad) for 1 h at room temperature followed by chemiluminescent detection (BioRad ChemiDoc). Antibodies used were TG2 polyclonal (PA5-95256; ThermoFisher) and GAPDH (1D4; NB300-221; Novus Biologicals). ImageLab (BioRad) was used for densitometry analysis.
9
Antibodies for Immunofluorescence and Immunoblotting
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Mouse monoclonal antibodies used in the study included those against PSF (for immunofluorescence, P2860, Sigma-Aldrich; and for immunoblotting, sc-101137, Santa Cruz), SC35 (S4045, Sigma-Aldrich), FLAG tag (F1804, Sigma-Aldrich) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, NB300-221, NOVUS). Rabbit polyclonal antibodies included those against CoAA (A300-331A, Bethyl), FLAG tag (F7425, Sigma-Aldrich) and RBM4 (2 (link)). The anti-hnRNP K rabbit monoclonal antibody was from Abcam (ab52600). Alexa Fluor 488- and 568-conjugated secondary antibodies were from Invitrogen.
10
Western Blot Analysis of Polycystins
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Cells were lysed in RIPA buffer boiled at 95 °C for 5 min. And after quantification loaded to 4-15% mini-Protean TGX gel (4568086, Bio-Rad) and transferred to PVDF membranes (162-0177, Bio-Rad). The membranes were blotted for polycystin-1, rabbit polyclonal antibody (1:2,000, ABT128, Millipore) or polycystin-2, mouse monoclonal antibody (1:1,000, sc-47734, Santa Cruz Biotechnology). GAPDH was used for loading control, mouse monoclonal antibody (1:2,000, NB300-221, NovusBio). For full details see Supplementary Methods .
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