Purified His6-MavC was used to raise rabbit specific antibodies using a standard protocol (Pocono Rabbit Farm & Laboratory). The antibodies were affinity purified as describe38 (link). For immunoblotting, samples resolved by SDS-PAGE were transferred onto 0.2 μm nitrocellulose membranes (Pall Life Sciences cat# 66485). Membranes were blocked with 5% non-fat milk, incubated with the appropriate primary antibodies: anti-UbE2N (Cell signaling, cat# 6999S, Thermo Fisher Scientific, cat# 37–1100), 1:1,000; anti-Flag (Sigma, Cat# F1804), 1: 2000; anti-HA (Santa Cruz, cat# sc-7392 1:1,000, Roche, cat# 11867423001 1:5,000), anti-ICDH39 (link), 1:10,000, anti-actin (Sigma, cat# A2103), 1:5,000, anti-tubulin (DSHB, E7) 1:10,000, anti-Ub K63 (Millipore, cat# 05–1308), 1:1,000, anti-IκBα (Cell signaling, cat# 9242S), 1:1,000, anti-Ub (Santa Cruz cat#sc-8017), 1:1,000, anti-p-IκBα (Cell Signaling, cat# 9246S), 1:1000, anti-UbE2K (Cell Signaling, cat# 8226S), 1:1000, anti-UbE2S (Cell Signaling, cat# 11878S), 1:1000, anti-UbE2E2 (Abcam, cat# Ab177485), 1:1000, anti-His (Sigma, cat# H1029), 1:10,000, anti-p65 (Cell signaling, cat# 8242S), 1:500. Membranes were then incubated with an appropriate IRDye infrared secondary antibody and scanned using an Odyssey infrared imaging system (Li-Cor’s Biosciences).
0.2 μm nitrocellulose membranes
Manufactured by Pall Corporation
The 0.2 μm nitrocellulose membranes are a laboratory filtration product designed for the retention of particles, microorganisms, and other microscopic materials. The membranes have a pore size of 0.2 microns, which makes them suitable for a variety of filtration applications in research and scientific settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti k63 ub, by Merck Group (2 mentions)
Anti tubulin, by Merck Group (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti p65, by Cell Signaling Technology (2 mentions)
Anti p iκbα, by Cell Signaling Technology (2 mentions)
Anti ub, by Santa Cruz Biotechnology (2 mentions)
Anti ube2n, by Cell Signaling Technology (2 mentions)
Anti ube2k, by Cell Signaling Technology (2 mentions)
Anti ube2s, by Cell Signaling Technology (2 mentions)
Anti ube2e2, by Abcam (2 mentions)
Anti ha, by Santa Cruz Biotechnology (2 mentions)
Anti iκbα, by Cell Signaling Technology (2 mentions)
Bca assay, by Merck Group (2 mentions)
Hrp conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Nb100 56875, by Novus Biologicals (1 mentions)
Proteinase inhibitor cocktail, by Roche (1 mentions)
Anti e cadherin 14472, by Cell Signaling Technology (1 mentions)
4 protocols using 0.2 μm nitrocellulose membranes
1
Immunoblotting of Ubiquitination Pathway Proteins
2
Immunoblotting of Ubiquitination Pathway Proteins
3
Western Blot Analysis of VGLL3 Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell lysates were isolated using cell lysis buffer (RIPA buffer: Cell Signaling Technology #9806, Danvers, MA) containing protease inhibitor cocktail (Roche, Nutley, NJ). Protein concentrations were determined by BCA assay (Sigma-Aldrich, St. Louis, MO). Proteins were separated by SDS-PAGE and transferred from gels to 0.2 μm nitrocellulose membranes (Pall Corporation, Washington, NY). The nitrocellulose membrane was further incubated overnight at 4°C with rabbit anti-VGLL3 (1:1000, Novus Biologicals, NB100-56875, Centennial, USA) and rabbit anti-GAPDH (1:2000, Novus Biologicals, 4650S, Centennial, USA). After that membranes were incubated with HRP-conjugated anti-Rabbit IgG (1:1000, Cell Signaling Technology, 7074S, Danvers, MA) secondary antibody for 1 hour at RT, protein bands were visualised using western blotting luminol reagent (Santa Cruz Biotechology, Inc., Dallas, Texas).
4
Western Blot Analysis of Epithelial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell lysates were isolated using cell lysis buffer (150 mM NaCl, 50 mM Tris pH 7.4, 1% NP-40, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM NaF, and 1 mM sodium pyrophosphate) containing proteinase inhibitor cocktail (Roche, Nutley, NJ). Protein concentrations were determined by BCA assay (Sigma-Aldrich, St. Louis, MO). Proteins were separated by SDS-PAGE and transferred from gels to 0.2 μm nitrocellulose membranes (Pall Corporation, Washington, NY). Protein bands were visualized using western blotting luminol reagent (Santa Cruz Biotechology, Inc., Dallas, Texas) after binding with a HRP-conjugated secondary antibody. Anti-Cytokeratin 7 (sc-23879), anti-Cytokeratin 18 (sc-515852), anti-EpCAM (sc-25308), anti-α-Actinin (sc-17829), and anti-GAPDH (sc-59541) antibodies were obtained from Santa Cruz Biotechnology, while anti-E-cadherin (#14472), anti-CD133 (#5860), and anti-CD44 (#3570) antibodies were purchased from Cell Signaling Technology (Danvers, MA)
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!