E1 and E2 enzymes and Ub were either purified in house or purchased from Boston Biochem, UBPBio, or Enzo Life Sciences. Ubiquitination reactions were incubated at 37°C in 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), or 1 mM β-mercaptoethanol and typically contained E1 (50 or 100 nM), E2 (280 nM Ubc13/Uev1A, 1 μM Ubc13, or 1 μM Ubc5b), E3 (200 nM TRIM25 or 5 μM RING), Ub (40 μM), ATP (3 mM), and MgCl2 (5 mM). Reactions were stopped by addition of SDS-PAGE sample buffer and boiling for 10 min. Typically, time points were taken at 0, 5, and/or 20 min. Immunoblots were performed with anti-Ub (1:2,000; P4D1; Santa Cruz Biotechnology), K63-linkage-specific anti-Ub (1:1,000; Enzo Life Sciences), anti-pentaHis (1:1,000; QIAGEN), and anti-FLAG M2 (1:5,000; Sigma). Signals were detected with a fluorescent secondary antibody (Rockland) and a LI-COR Odyssey scanner.
Anti penta his
Manufactured by Qiagen
Sourced in Germany
The Anti-Penta-His is a lab equipment product that is used for the detection and purification of recombinant proteins containing a histidine tag. It is designed to bind specifically to the histidine tag, allowing for the isolation and identification of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2, by Merck Group (8 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (6 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Fluorescent secondary antibody, by LI COR (2 mentions)
K63 linkage specific anti ub, by Enzo Life Sciences (2 mentions)
Odyssey scanner, by LI COR (2 mentions)
Nhs fitc, by Thermo Fisher Scientific (2 mentions)
Oti1e4, by OriGene (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti kss1, by Santa Cruz Biotechnology (2 mentions)
Odyssey clx imager, by LI COR (2 mentions)
Nupage system, by Thermo Fisher Scientific (1 mentions)
Anti s6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Anti tom20, by Santa Cruz Biotechnology (1 mentions)
Anti tfam, by Santa Cruz Biotechnology (1 mentions)
Anti ubf1, by Santa Cruz Biotechnology (1 mentions)
Anti fibrillarin, by Santa Cruz Biotechnology (1 mentions)
Ab16048, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated anti rabbit ig, by Agilent Technologies (1 mentions)
Anti ha 11 clone 16b12, by Fortrea (1 mentions)
21 protocols using anti penta his
1
Ubiquitination Reaction Protocols
2
Immunoblotting of Subcellular Fractions
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Immunoblotting was performed with whole cell lysates, fractionated lysates, isolated nuclei or immunoprecipitation samples using the NuPAGE system according to the manufacturer's recommendations (Invitrogen). For preparation of whole cell lysates cells were washed with PBS and lysed in Laemmli lysis buffer (67 mM Tris-HCl pH 6.8, 0.7% SDS). Subcellular fractionation was performed using the ProteoExtrac Subcellular Proteome Extraction Kit (Calbiochem) following the manufacturer's protocol. Immunoprecipitations were performed using standard protocols. The following antibodies were used: anti-penta His (Qiagen), anti-FlagM2 (Sigma), OXPHOS complex I subunit 39 k (Invitrogen), anti-S6 ribosomal protein (Cell Signalling), anti-Tfam, anti-Tom20, anti-Ubf1, anti-Fibrillarin (Santa Cruz Biotechnology). The Alexafluor 680-conjugated secondary antibodies from different species were purchased from Invitrogen. The IRD-800 antibodies were purchased from Rockland. The polyclonal antibody against NOA1 was raised in rabbits by Eurogentec (Liege, Belgium) and detects specifically precursor and mature NOA1 protein (Figure S1 in File S1 ).
3
Western Blotting and PAR-CLIP Antibody Validation
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Monoclonal anti-HA.11 (clone 16B12, Covance), anti-β-tubulin (Sigma, SAB3500350, T0198), anti-lamin B1 (Abcam Ab16048), anti-HuR (Santa Cruz [19F12] sc-56709), anti-Penta-His (Qiagen 34660) were used as primary antibodies at 1:1000 dilution. HRP-conjugated anti-rabbit Ig and anti-mouse Ig (both from DAKO) were used as secondary antibodies at 1:4000 dilution for Western Blotting analysis. Anti-Flag M2 (Sigma, F3165) was used for PAR-CLIP. We evaluated the following commercial anti-RBPMS antibodies, Santa Cruz S-25, Santa Cruz E13, Abnova 8390, Sigma AV40269, using recombinant protein and overexpressing HEK293 cell lysates as controls; none of these antibodies were sensitive and specific in detecting human RBPMS or RBPMS2. We obtained a rabbit polyclonal antibody for human RBPMS using recombinant protein, and were able to specifically detect 1 ng of recombinant RBPMS at 1:100 dilution; however, the low expression levels of RBPMS in HEK293 cells did not allow detection of endogenous protein.
4
Western Blot Protein Detection
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To detect specific proteins, they were separated on a polyacrylamide gel and blotted onto a PVDF-membrane (Bio-Rad Laboratories, Munich, Germany) with the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Munich, Germany) according to the manufacturer's instructions. The membrane was incubated with blocking solution (3% (w/v) bovine serum albumin (BSA) in TBS for 1 h. Subsequently, the primary antibody anti-Penta-His (1:2,000) (Qiagen, Hilden, Germany) was added and incubated over night at 4°C under gentle shaking. On the next day, the membrane was washed three times with T-TBS for 10 min. The secondary antibody anti-mouse-IgG-HRP was added to the membrane and incubated for 1 h followed by triple washing with T-TBS for 10 min. Bands could be visualized by addition of a luminol containing substrate (Immobilon Western HRP Substrat, Merck, Darmstadt, Germany). Chemiluminescence was measured with ChemiDoc XRS+ system (Bio-Rad Laboratories, Munich, Germany).
5
Antibody Sources and Reagents for Cellular Studies
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The primary antibody against SNX9 (OTI1E4, 1:1000) was purchased from Origene, anti-Pacsin (PA5-83983, 1:200), anti-N-WASP (PA5-52198, 1:200) and anti-β-actin (MA5-15739, 1:2000) antibodies were sourced from Thermo Scientific and the anti-actin (#7301-01, 1:500) antibody was from Hypermol. Anti-penta-His (#34660, 1:2500) and anti-GST (#2622, 1:1000) antibodies were obtained from Qiagen and Cell Signaling, respectively. Antibodies against Cpn0677 were generated by Eurogentec (Belgium, 1:50 in immunofluorescence).
Secondary anti-rabbit, anti-rat and anti-mouse antibodies coupled to Alexa488 or Alexa594 (2 µg/ml) or coupled to alkaline phosphatase (1:10,000) were purchased from Thermo Scientific. Rhodamine-Phalloidin (#R415) was purchased from Thermo Scientific. All lipids used in this study were obtained from Avanti Lipids, NHS-FITC and NHS-Rhodamine and MitoTracker™-Red from Thermo Scientific. Wiskostatin (W2270-5MG) was purchased from Merck. G-actin (#8101-01), G-actin-Atto647 (#8158-03) and Arp2/3 (#8413-01) were obtained from Hypermol.
Secondary anti-rabbit, anti-rat and anti-mouse antibodies coupled to Alexa488 or Alexa594 (2 µg/ml) or coupled to alkaline phosphatase (1:10,000) were purchased from Thermo Scientific. Rhodamine-Phalloidin (#R415) was purchased from Thermo Scientific. All lipids used in this study were obtained from Avanti Lipids, NHS-FITC and NHS-Rhodamine and MitoTracker™-Red from Thermo Scientific. Wiskostatin (W2270-5MG) was purchased from Merck. G-actin (#8101-01), G-actin-Atto647 (#8158-03) and Arp2/3 (#8413-01) were obtained from Hypermol.
6
Antibody Panel for Btk Signaling
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Anti-Total pY (clone 4G10), 1:1000, Millipore # 05-321; anti-Btk (D6T2C), 1:1000, Cell Signaling # 56044; anti-Btk, 1:1000, Thermo Scientific # PA5-27392; anti-Btk (pY551)/Itk (pY511) Clone 24a, 1:1000, BD Biosciences # 558034; anti-Btk (pY223), 1:1000, Cell Signaling #5082; anti-p44/42 MAPK (Erk1/2), 1:1000, Cell Signaling #9102; anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10), 1:1000, Cell Signaling #9106; anti-PLCƴ2, dilution 1:1000, Cell Signaling, #3872; anti-PLCƴ2 (Tyr1217), 1:1000, Cell Signaling #3871; anti-Flag, 1:5000, Sigma # F3165; anti-Penta-his, 1:5000, Qiagen #34660; anti-PLCƴ2 (Tyr753) [EPR5914-3], 1:1000, Abcam #ab133455; anti-Tubulin, 1:2000, Sigma #T9026; anti-Myc-tag Myc.A7 DyLight800, 1:10,000, Thermo Scientific #MA1-21316-D800; anti-mouse IgG IRDye 800CW, 1:10,000, LiCor #926-32210; anti-Rabbit IgG (H+L) IRDye800, 1:10,000, Rockland #611-732-127; anti-Mouse IgG (H+L) Peroxidase AffiniPure, 1:10,000, Jackson ImmunoResearch #115-035-003; anti-Rabbit IgG (H+L) Peroxidase AffiniPure, 1:10,000, Jackson ImmunoResearch #111-035-003. The full information on primary and secondary antibodies that were used for the reported experiments is available in the Reporting Summary.
7
In vivo Protein Cross-linking Assay
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For in vivo photo-crosslinking experiments, equivalent amounts of protein were loaded into each lane according to the OD600 of the cultures. Rabbit anti-LolC, -LolE and -Pal antisera were described previously (17 (link)). The antiserum against LolE used in this study also reacts with Pal (15 (link)), and usually yields good results in the detection of LolC–Pal cross-link products. Therefore, in some experiments, anti-LolE was used to detect the LolC–Pal cross-linking products. Anti-pentaHis was obtained from Qiagen.
8
Immunoblotting of Bacterial Proteins
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Protein lysates for immunoblotting were prepared by using Laemmli sample buffer. Protein lysates corresponding to equal OD600 were loaded on 4–12% Bis/Tris gels (Invitrogen), and run in TGS buffer. Protein transfer was performed with iBlot gel transfer stacks (Invitrogen). Membranes were probed with the following monoclonal antibodies: mouse anti-PdpB, mouse anti-IglC, and mouse anti-IglB (all provided from BEI Resources, Manassas, VA, USA) or via commercially available anti-HA (clone HA-7, Sigma), anti-Penta-His (Qiagen, MD, USA). A secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Santa Cruz Biotechnology, CA, USA) and the Enhanced Chemiluminescence system (ECL) (Amersham Biosciences, Uppsala, Sweden) were used.
9
Quantifying Cx26 Protein Expression
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Cx26 expression was assessed from Western blots of cell lysates using antibodies against the histidine–tag (anti-Penta-His, Qiagen) or the Cx26 intracellular loop region (Life Technologies). Detection in Western blots was by imaging (Odyssey Infrared Imager, Li-Cor Biosciences) of goat anti-rabbit IRDye 800 (Li-Cor Biosciences) for the anti-Cx26 antibodies or goat anti-mouse Alexa Fluor 680 (Life Technologies) for the anti-histidine antibody.
10
Antibody Sources and Reagents for Protein Analysis
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The primary antibody against SNX9 (OTI1E4) was purchased from Origene, anti-EGFR (PA1-1110) and anti-GFP (MA5-15256) were from Thermo Scientific, antipenta-His (#34660) from Qiagen, anti-GST (#2622) from Cell Signaling, and anti-DsRed (sc-101526) from Santa Cruz. Anti-IncA was a gift from G. Zhong, University of Texas Health Science Center at San Antonio, San Antonio, TX (57 (link)), and anti-DnaK was obtained from S. Birkelund, Aalborg University, Aalborg, Denmark (58 (link)). Antibodies against Cpn0677 and Cpn0678 were generated in our laboratory, as was anti-CPn0147. Mouse anti-Cya, rabbit anti-CRP, and rabbit anti-IpaD antibodies were generously donated by N. Guiso, A. Ullmann, and C. Parsot, Institut Pasteur, Paris, France, respectively. Secondary anti-rabbit and anti-mouse antibodies coupled to Alexa488 or Alexa594 were purchased from Thermo Scientific, and those coupled to alkaline phosphatase were sourced from Promega. CellMask (orange) and Rhodamine-Phalloidin were purchased from Thermo Scientific, and SiR-Tubulin from Spirochrome. All lipids used in this study were obtained from Avanti Lipids, and Texas red dye, NHS-FITC and DyLight650NHS from Thermo Scientific. Nocodazole and Cytochalasin D were purchased from Merck.
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