The primary antibodies used were as follows: Actin (Sigma‐Aldrich, SAB5500001), ATG5 (Nanotools, clone 7C6), DAXX (Santa‐Cruz, clone M‐112), Flag (Sigma‐Aldrich, clone M2), GAPDH (Cell Signaling Technology, #2118), GFP (Cell Signaling Technology, #2956), HA (Abcam, ab9110), Halo (Promega, G9211), His (Santa‐Cruz, clone H‐3), HMOX‐1 (Abcam, ab137749), Keap1 (Proteintech, 10503‐2‐AP), LAMP1 (Cell Signaling Technology, #9091), LC3B (Cell Signaling Technology, #2775), MOAP‐1 (Sigma‐Aldrich, HPA000939), Myc (Santa‐Cruz, clone 9E10), NBR1 (Abnova, clone 6B11), Nrf2 (MBL International, clone 1F2), p62/SQSTM1 (Abcam, ab56416), TAK1 (Abcam, ab109526), Tom20 (Santa‐Cruz, clone F‐10), TRIM21 (Proteintech, 12108‐1‐AP), Tubulin (Santa‐Cruz, clone TU‐02), Ubiquitin (Santa‐Cruz, clone P4D1), Ubiquitin (linkage‐specific K63) (Abcam, ab179434), Ubiquitin (linkage‐specific K48) (Abcam, ab140601).
Ubiquitin
Manufactured by Abcam
Sourced in United States, United Kingdom
Ubiquitin is a small regulatory protein that is found in all eukaryotic cells. It is involved in the tagging of proteins for degradation by the proteasome, a process known as ubiquitination. Ubiquitin can be used in a variety of laboratory applications, such as the study of protein degradation pathways and the investigation of cellular processes regulated by ubiquitination.
Automatically generated - may contain errors
Lab products found in correlation
Flag tag (flag), by Merck Group (6 mentions)
β actin, by Santa Cruz Biotechnology (6 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
β tubulin, by Cell Signaling Technology (3 mentions)
Hif 2α, by Novus Biologicals (3 mentions)
Ab7780, by Abcam (3 mentions)
β tubulin, by Santa Cruz Biotechnology (3 mentions)
E cadherin, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (3 mentions)
Lamin a, by Santa Cruz Biotechnology (2 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Bcl 2, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ubiquitin, by Santa Cruz Biotechnology (2 mentions)
Sqstm1 p62, by Abcam (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Rna pol 2, by Abcam (2 mentions)
54 protocols using ubiquitin
1
Comprehensive Antibody Characterization for Cellular Analyses
2
Western Blot Analysis of Protein Expression
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Protein samples were prepared in reduced and denatured forms (32 (link)) and resolved using SDS-PAGE. The proteins were transferred to a nitrocellulose membrane and then blocked with 5% nonfat milk in TBS-T (0.02 M Tris–HCl, 0.16 M NaCl, and 0.1% Tween-20, pH 7.4), at room temperature, for 1 h. The membranes were incubated overnight with primary antibodies PABPC4 (Bethyl, #A301-466A), NCoR1 (Affinity, #AF0270), PABPC1 (Thermo Fisher Scientific, #PA5-29883), FLAG (Sigma-Aldrich, #F1804), α-tubulin (Sigma-Aldrich, #T9026), OXPHOS (proteins of mitochondrial ETC) (Abcam, #ab110413), PPARD (Thermo Fisher Scientific, #PA1-823A), eIF4G (Cell Signaling Technology, #2498), puromycin (Merck, #MABE343), Vinculin (Cell Signaling Technology, #4650), Lamin A (Santa Cruz Biotechnology, #sc-71481), and β actin (Santa Cruz Biotechnology, #81178), Akt (Cell Signaling Technology, #9272), p-AktThr308 (Cell Signaling Technology, #9275), ubiquitin (Abcam, #ab7254), and GST (Sigma-Aldrich, #G7781). The membrane was then washed with TBS-T and incubated with horseradish peroxidase–conjugated secondary antibody (1:10,000) in TBS-T solution containing 5% nonfat for 1 h. Membranes were washed with TBS-T and then added the peroxidase substrate SuperSignal West Plus (Thermo Fisher Scientific), and the band intensities were captured in the ImageQuant LAS500 (GE Healthcare).
3
SDS-PAGE Immunoblotting Protocol
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Cells were lysed in lysis buffer (SDS 1%, Tris pH 7.4 10 mM, Sodium orthovanadate 1 mM). Samples in Laemmli buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol blue) containing equal amounts of total protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to PVDF membranes (Thermo Scientific, Waltham, MA, USA), membranes were probed with primary antibodies PARP, β-tubulin (Cell signaling, Leiden, The Netherland) or ubiquitin (Abcam, Paris, France). Membranes were then probed with secondary fluorescent antibodies (Li-COR IRDye). Antibody binding was visualized with the LI-COR odyssey Fc system (Cambridge, UK).
4
HIF1α Immunoprecipitation and Immunoblot
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Cell pellets were lysed using a RIPA lysis buffer with Halt proteasome inhibitor (Thermo Scientific), sonicated briefly and centrifuged at 15000 × g at 4°C for 15 minutes. Immunoprecipitation was performed as described previously [55 (link)]. Antibodies against HA-HIF1α were obtained from Covance (Princeton, New Jersey). An indirect immunoprecipitation kit (Millipore) and manufacturer-suggested protocols were used. Immunoblot was performed as described previously [56 (link)]. PVDF Membranes were probed using antibodies to HA-HIF1α (Covance), HO-HIF1α (Cell Signalling Technology, Danvers, Massachusetts), HIF1α (Novus Biologicals, Littleton, Colorado), HIF2α (Novus) or ubiquitin (Abcam, Cambridge, Massachusetts).
5
Western Blot Analysis of Protein Expression
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Equal amounts of each protein diluted in 5 × sodium dodecyl sulfate (SDS) loading buffer (Beyotime, Shanghai, China) were separated using SDS–polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). After being blocked at room temperature for 1 h, the membranes were incubated with primary antibodies against GAPDH (1:1000, Cell Signaling Technology, Massachusetts, USA, 5174), p21 (1:1000, Abcam, Cambridge, UK, 109,520), p53 (1:1000, Cell Signaling Technology, 48,818), p16 (1:1000, Abcam, 51,243), SERPINE2 (1:1000, Abcam, 134,905), YBX3 (1:2000, Bethyl, Montgomery, USA, A303-070A), β-Tubulin (1:1000, Cell Signaling Technology, 2128), PCNA (1:1000, Abcam, 29), Histone3 (1:1000, Abcam, 176,842), ubiquitin (1:1000, Abcam, 134,953), HNRNPC (1:1000, Abcam, 133,607), YBX1 (1:1000, Proteintech, Rosemont, USA, 20,339–1-AP), and ZO-1 (1:1000, Proteintech, 21,773–1-AP) overnight at 4 °C. The membranes were washed 3 times, incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000, BOSTER, California, USA, BA1050, BA1054) for 1 h at room temperature and then washed 3 times with TBST. Specific immunoreactive bands were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The mean intensity ratio was analyzed by ImageJ 1.4.
6
Protein Expression Analysis of TRIM69 and Apoptosis Markers
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The specimens and HLECs were lysed with RIPA lysis buffer at 4 °C. A bicinchoninic acid assay kit (Sigma Chemical Co., St. Louis, MO) was used for protein quantification. Proteins of each sample (25 μg per sample) were subjected to 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot assays were performed with primary antibodies to the following: TRIM69 (Ab111943; Abcam, Cambridge, MA, USA); p53 (Ab26; Abcam); Bax (Ab32503; Abcam); Bcl-2 (Ab32124; Abcam); Foxo3a (Ab53287; Abcam); ubiquitin (Ab7780; Abcam); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#5174; Cell Signaling Technology, Danvers, MA, USA). GAPDH protein was used as the loading control.
7
Western Blot Analysis of Protein Expression
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Total proteins were obtained from HCC cells using RIPA buffer (Beyotime, Shanghai, China) and quantified by Enhanced BCA Protein Assay Kit (Beyotime). Then, 20 μg proteins were undergoing SDS-PAGE and transferred to the PVDF membrane (Millipore, Billerica, MA, United States). After blocking, the membrane was incubated overnight with primary antibodies at 4°C. The next day, secondary antibody incubation was performed at room temperature for 1–2 h. Enhanced chemiluminescence (ECL) reagent (Millipore) was used for luminous reaction and western blot was photographed by Amersham Imager 680 (GE Healthcare Life Sciences, Pittsburgh, PA, United States). The used primary antibodies: HIF-1α (ab1, Abcam, Cambridge, MA, United States), HIF-2α (ab199, Abcam), RNF146 (ab201212, Abcam), β-tubulin (10094-1-AP, Proteintech, Wuhan, China), PTEN (ab267787, Abcam). p-AKT (Ser473, 66444-1-Ig, Proteintech), AKT (60203-2-Ig, Proteintech), p-mTOR (Ser2448, 67778-1-Ig, Proteintech), mTOR (66888-1-Ig, Proteintech), Ubiquitin (ab7254, Abcam).
8
Protein Fractionation and Immunoblotting
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The total protein extract was prepared by homogenizing the cells in a 1× SDS sample buffer. The NP-40-soluble and -insoluble protein fractions from the cells were prepared as previously described [34 (link)]. Immunoprecipitation and western blot were carried out as previously described [34 (link)]. Primary antibodies against the following proteins were used for western blot analysis: K48-linked Ub chain-specific antibody (Millipore, 05-1307); K63-linked Ub chain-specific antibody (Abcam, ab179434); ubiquitin (Abcam, ab134953); Phospho-SQSTM1 (Thr269/Ser272)-specific antibody (Phosphosolutions, P196-269); SQSTM1 (Santa Cruz Biotechnology (sc-28359); ATG5 (Cell Signaling Technology, 12994); LC3B (Cell Signaling Technology, 3868); ATG16L1 (Abcam, ab187671); WIPI2 (Abcam, ab105459); Beclin1 (Proteintech, 11306-1-AP); GFP (Rockland, 600-101-215); FLAG tag (Prospec, ANT-146-b); Myc Tag (Biolegend, MMS-150R); GAPDH (Zen Bioscience, 200306); β-actin (Zen Bioscience, 200068-6D7). See Supplementary Table S2 for further details and dilutions of all antibodies.
9
Immunoblotting Analysis of NDRG1 and Ubiquitin
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MCF-7 cells were lysed in RIPA lysis buffer (Sigma), and protein concentration was determined using Bio-Rad Protein assay reagent (Bio-Rad Laboratories, Hercules, USA). Then, the protein lysate was separated by 10% SDS-PAGE and transferred to a PVDF membrane (Bio-Rad Laboratories). The membranes were blocked by TBST Blocking buffer (Arrowtec, Taiwan) for 10 min and hybridized to a primary antibody consisting of NDRG1 (Abcam Inc., Cambridge, USA) and ubiquitin (Abcam). After immunoblotting, the membranes were washed by TBS (Omics Bio, Taiwan) with Tween20 and reacted with horseradish peroxidase-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG (GeneTex, Irvine, USA). The protein bands were visualized using an enhanced chemiluminescence system (Millipore, Billerica, USA).
10
Protein Lysate Immunoblotting Protocol
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Protein lysates were prepared from E10.5 dissected limb buds or embryos or cultured MEFs using Dignam buffer. 50 ug of total protein were then subjected to SDS-PAGE analysis followed by immunoblotting according to standard protocols. Primary antibodies: Sufu (#2522, Cell signaling), Spop (#PA5-28522), Myc (#SC-789, Santa Cruz), Flag (#F7425, Sigma), E2F1 (#137415, Abcam), Ubiquitin (#7780, Abcam), Gli3 (AF3690, R and D systems), Tbx3 C-terminal antibody (Frank et al., 2013 (link)); Kif7 (ab 95884, Abcam); β tubulin (Santa Cruz).
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