Western blotting was performed as previously described.8 (link) Primary antibodies were used (1:1,000 in 5% milk): NDUFA4L2 (Abcam ab74138, rabbit polyclonal), NDUFA4L2 (Proteintech 66050-1-Ig, mouse monoclonal), FLAG (GenScript, A00187S, mouse monoclonal). Secondary antibodies: anti-Rabbit IgG (Jackson, 711–135-052, 1:10,000 diluted in 5% milk), anti-Mouse IgG (Jackson, 715–035-150, 1:10,000 diluted in 5% milk).
Flag tag (flag)
Manufactured by GenScript
Sourced in United States
FLAG is a sequence-specific peptide epitope tag used for protein identification and purification. It facilitates the detection and isolation of recombinant proteins expressed in various cell systems.
Automatically generated - may contain errors
Lab products found in correlation
Ndufa4l2, by Proteintech (1 mentions)
Ndufa4l2, by Abcam (1 mentions)
Irdye 800 secondary antibody, by LI COR (1 mentions)
Pex19, by Novus Biologicals (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Nfat5, by Santa Cruz Biotechnology (1 mentions)
β tubulin, by Proteintech (1 mentions)
Pdcd4, by Santa Cruz Biotechnology (1 mentions)
10094 1 ap, by Proteintech (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
β actin, by Proteintech (1 mentions)
Radioimmunoprecipitation lysis buffer, by Beyotime (1 mentions)
P akt thr308, by Cell Signaling Technology (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
T hsl, by Cell Signaling Technology (1 mentions)
P hsl ser563, by Cell Signaling Technology (1 mentions)
Protease phosphatase inhibitor cocktail, by Roche (1 mentions)
T akt, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
6 protocols using flag tag (flag)
1
Quantitative Protein Analysis via Western Blot
2
PEX19 and PEX26 Crosslinking Assay
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400 nM PEX19Bpa was incubated without and with 100 nM PEX26 at room temperature for 1 min. The reaction was further incubated with 400 nM PEX3ΔN-WT and its variants at room temperature for 5 min. The reaction was UV-irradiated as described in the PEX26Bpa crosslinking assay. The crosslinked samples were loaded onto 8% Tris-glycine gels and analyzed by western blotting using Strep (1:3000 dilution, Genscript), PEX19 (1:3000 dilution, Novus Biologicals), Flag (1:3000 dilution, Genscript), and IRDye800 secondary antibodies (1:20,000 dilution, LiCor).
3
Western Blot Protein Expression Analysis
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Cells were lysed with cold RIPA buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1 mmol/L EDTA) supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany) and quantified with the bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (~ 30 μg) were subjected to SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skimmed milk in TBST (20 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, and 0.1% Tween 20), membranes were incubated with primary antibodies overnight at 4 °C and then with HRP-conjugated secondary antibodies. The protein bands were visualized with the chemiluminescent detection module (Pierce Biotechnology, Rockford, IL, USA) and images were taken with the Tanon-5200 imaging system (Tanon, Shanghai, China). The following primary antibodies were used: PDCD4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, SC-13054, 1:1,000), NFAT5 (Santa Cruz Biotechnology, SC-13035, 1:200), FLAG (GenScript, Piscataway, NJ, USA, A00013, 1:1,000), β-actin (Proteintech, Wuhan, Hubei, China, 66009–1-Ig, 1:10,000;), and β-tubulin (Proteintech, 10094-I-AP, 1:5,000).
4
Western Blot Analysis of Cellular Proteins
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Tissue was homogenized, and protein was extracted using radioimmunoprecipitation lysis buffer (Beyotime) containing phosphatase/protease inhibitor cocktail (Roche). Protein concentration was measured using the BCA protein assay kit (Beyotime). Equivalent amount of protein was loaded onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto a nitrocellulose membrane (MilliporeSigma). The membrane was blocked with 5% skim milk for 1 h at room temperature, and incubated overnight at 4 °C with primary antibody against CLSTN3 (Proteintech, 13302-1-AP), CAV1 (Proteintech, 66067-1-Ig), Actin (Abcam, ab8227), Tubulin (MilliporeSigma, T6199), UCP1 (Abcam, ab10983), P-AKTThr308 (Cell Signaling Technology, 13,038), T-AKT, (Cell Signaling Technology, 9272), P-HSLSer563 (Cell Signaling Technology, 4139), T-HSL (Cell Signaling Technology, 4107), FLAG (GenScript, AF519), HA (Cell Signaling Technology, 3724), APP (GeneTex, GTX101336), VDAC1 (Abcam, ab154856), and OXPHOS rodent WB cocktail (Abcam, ab110413). Then, the membrane was incubated with secondary anti-rabbit or anti-mouse antibody for 1 h at room temperature. Protein bands were detected with ECL chemiluminescent reagent (MilliporeSigma) using the Image Quant LAS4000 System (GE Healthcare).
5
Androgen Receptor Regulation in Prostate Cancer
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Primary antibodies; Androgen receptor (AR, cat#3202S), tubulin (cat#9F3), Lamin A/C (cat#2032), and HA-TAG (cat#3724S) primary antibodies (Cell Signaling Technology, Danvers, MA, USA), NRDP1 (Santa Cruz Biotechnology, Dallas, TX, USA, cat#sc-365622), GAPDH (Sigma-Aldrich, St. Louis, MO, USA, cat#39-8600), FLAG (Genscript, Piscataway, NJ, USA, cat#A00187-200). Secondary antibodies; HRP-linked anti-mouse IgG (Cell Signaling Technology, Danvers, MA, USA, cat#7076), HRP-linked goat anti-rabbit secondary (Jackson Immunoresearch, West Grove, PA, USA, cat#111-035-144). siRNA; AR siRNA was a pool of 4 duplexes with the following sequences; #1: 5′-CAGUCCCACUUGUGUCAAATT-3′, #2: 5′-CUGAUCUGUGGAGAUGAATT-3′, #3: 5′-GUCGUCUUCGGAAAUGUUATT-3′, #4: 5′-GACAGUGUCACACAUUGAATT-3′ (Santa Cruz Biotechnology, Dallas, TX, USA). Control siRNA was a pool of 4 scrambled non-specific siRNA duplexes (Santa Cruz Biotechnology, Dallas, TX, USA). Drugs; R1881 (Sigma-Aldrich, St. Louis, MO, USA), Enzalutamide, Cycloheximide, MG132 (Selleckchem, Houston, TX, USA).
6
Westerns for Protein Detection
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Cells were lysed in RIPA lysis buffer. Nuclear extracts were prepared with Nuclear Extract Kits (Active Motif, Carlsbad, CA, United States). Total cell lysates or nuclear and cytosol protein extracts were separated by 10% SDS–PAGE and blotted onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were immunoblotted with antibodies against the following: SMAD4 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, United States), MyD88 (1:1000) (Cell Signaling, Danvers, MA, United States), phosphorylated SMAD5 (1:1000) (Abcam, Cambridge, MA, United States), SMAD1 (1:1000) (Cell Signaling), His (1:1000) (Genscript, Piscataway, NJ, United States), Flag (1:5000) (Genscript), HA (1:5000) (Genscript), Ubiquitin FK2 (1:100), Ferroportin 1 (1:1000) (Novus Biologicals, Littleton, CO, United States), GST (1:1000) (Genscript), and β-actin (1:10000) (Abcam, Cambridge, MA, United States). For secondary antibodies, anti–rabbit IgG (1:5000) or anti–mouse IgG (1:5000) were used. Antigen-antibody complexes were visualized with the ECL Western Blotting Detection Reagent (Invitrogen).
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