Estradiol (E2) was purchased from Sigma-Aldrich. Antibodies against the following epitopes were used: Myc (9E10) and HA (12CA5) from Roche Applied Science (Germany), GFP (G1544) from Santa Cruz Biotechnology (USA), and α-Tubulin (B-5-1-2) from Sigma-Aldrich (USA).
Ha 12ca5
Manufactured by Roche
Sourced in United States
HA (12CA5) is a monoclonal antibody produced by Roche. It recognizes the HA-tag, a commonly used protein tag for recombinant protein expression and purification. The core function of HA (12CA5) is to specifically bind to the HA-tag, enabling detection and isolation of HA-tagged proteins in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Flag m2, by Merck Group (4 mentions)
α tubulin b 5 1 2, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ubiquitin, by Zymo Research (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Standard enhanced chemiluminescence reagents, by GE Healthcare (2 mentions)
Horseradish peroxidase conjugated secondary antibodies mouse and rabbit, by Merck Group (2 mentions)
Gapdh 6c5, by Santa Cruz Biotechnology (2 mentions)
Hif1α h1α67, by Santa Cruz Biotechnology (2 mentions)
Nedd8 a 812, by R&D Systems (2 mentions)
Gst b 14, by Santa Cruz Biotechnology (2 mentions)
P53 do 1, by Santa Cruz Biotechnology (2 mentions)
Estradiol e2, by Merck Group (1 mentions)
Af6000, by Leica (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa flour 488, by Thermo Fisher Scientific (1 mentions)
E cadherin, by BD (1 mentions)
β catenin, by Merck Group (1 mentions)
T7 tag, by Merck Group (1 mentions)
19 protocols using ha 12ca5
1
Antibody Validation Protocol
2
Immunostaining of Cytoskeletal Proteins
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Cells were fixed in 4% PFA, washed and blocked in 1%BSA/0.1% Tween 20/PBS and incubated with a primary antibody against human αSMA (Human alpha-Smooth Muscle Actin Alexa Fluor® 488-conjugated Antibody, R&D systems), HA (12CA5, Roche) or ALK4 (Activin A Receptor Type IB/ALK-4, Abcam). Then, cells were incubated with a secondary antibody (Alexa Fluor 488, 555 or 647, Thermo Scientific) combined with phalloidin conjugated antibody (Rhodamine Phalloidin, Invitrogen). Lastly, cells were stained with DAPI (Thermo Scientific). Imaging was performed using the Leica AF6000.
3
Antibody Generation and Characterization
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A rabbit polyclonal antibody for hCRB1 was raised against a GST fusion protein corresponding to amino acids 255–407 within the extracellular domain of the protein. hMIB1 and mEPB41L5 polyclonal antibodies were raised against GST fusion proteins corresponding to amino acids 236–445 and 669–732 of the proteins respectively, and antisera affinity purified using the corresponding antigen. Additional primary antibodies used available from commercial sources include: HA12CA5 (Roche,IF 1:500, IB 1:1000), HA-Tag (C29F4; Cell Signaling Technology 3724; IP 5μl), Histidine (HIS.H8, Sigma-Aldrich 05-949, IB 1:500), FLAGM2 (Sigma-Aldrich, mouse, IF 1:500, IB 1:1000; Abnova, rabbit, IF: 1:500), myc9E10 (Clontech,; IF 1:500, IB 1:1000), T7tag (EMD Millipore 69522-3; IB 1:500) E-cadherin (BD Biosciences, mouse, IF 1:300), ZO1 (Zymed, mouse, IF 1:300; Millipore, rat, IF 1:500), β-catenin (Millipore, rabbit, IF 1:300), Actin-phalloidin (Molecular Probes, Invitrogen, IF 1:500), GFP (ThermoFisher Scientific A-11122; IF 1:500) and CRB3 (Sigma HPA013835, IF 1:50). Secondary antibodies conjugated with AlexaFlour 488 (Molecular Probes), Cy3 and Cy5 (Jackson Laboratories).
4
Lentiviral Nedd4-1 shRNA Clones
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Lentiviral human Nedd4-1 shRNA clones were purchased from Open Biosystems. The mAb against the hemagglutinin (HA) (12CA5) was purchased from Roche Applied Science. The polyclonal anti-Nedd4-1 antibody was made by injecting GST-human Nedd4-1 fusion protein into rabbits and purified by protein A beads. IHC staining S-P kit (KIT-9710) was purchased from MAIXIN Biology Corporation. The gastric cancer cell lines AGS and N87 were purchased from ATCC.
5
Cell Culture and Transfection Protocol
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Human 293T, HCT116, SW480, and U2OS cells were cultured with Dulbecco's modified Eagle's medium media with 10% fetal bovine serum and antimicrobials. Human HBL1, DLD1, and BT483 cells were maintained in RPMI‐1640 medium supplemented with fetal bovine serum and antimicrobials. For transient transfection, cells were transfected with DNA using Lipofectamine 2000 (Invitrogen). Antibodies to the following epitopes and proteins were purchased from the indicated vendors: CSN6 (Enzo Life Sciences), HA (12CA5, Roche and Proteintech), ubiquitin (Zymed Laboratories), FOXO4 (Cell Signaling Technology and Santa Cruz Biotechnology) and COP1 (Bethyl Laboratories and Santa Cruz Biotechnology). Flag (M2 monoclonal antibody), PSAT1 and SHMT2 (Affinity Biosciences). PHGDH and Actin were purchased from Sigma. pPKB/Akt, PKB/Akt, and Glut1 were purchased from Cell Signaling Technology. GFP, MDM2, and Myc (mouse monoclonal 9E10) were purchased from Santa Cruz Biotechnology.
6
Protein Extraction and Western Blot
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Approximately 10 OD600 cells were collected from respective cultures, pelleted, and flash-frozen in liquid nitrogen until further use. The cells were resuspended in 400 μl of 10% trichloroacetic acid and lysed by bead-beating three times (30 s of beating and then 1 min of cooling on ice). The precipitates were collected by centrifugation, resuspended in 400 μl of SDS–glycerol buffer (7.3% SDS, 29.1% glycerol, and 83.3 mM Tris base), and heated at 100°C for 10 min. The supernatant after centrifugation was treated as the crude extract. Protein concentrations from extracts were estimated using bicinchoninic acid assay (Thermo Scientific). Equal amounts of samples were resolved on 4 to 12% Bis–Tris gels (Invitrogen). Coomassie blue–stained gels were used as loading controls. Western blots were developed using the antibodies against the respective tags. We used the following primary antibodies: monoclonal FLAG M2 (Sigma) and HA (12CA5, Roche). Horseradish peroxidase–conjugated secondary antibodies (mouse and rabbit) were obtained from Sigma. For Western blotting, standard enhanced chemiluminescence reagents (GE Healthcare) were used. ImageJ was used for quantification.
7
Western Blot Analysis of HPV Proteins
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Twenty micrograms of total protein was resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride membrane (GE Healthcare, Chicago, IL, USA). The membrane was then probed with specified primary antibodies overnight as follows: HPV16 E7 (Cervimax, Vienna, Austria), β‐galactosidase (Promega, Madison, WI, USA), HA (12CA5; Roche Diagnostics, Risch‐Rotkreuz, Switzerland), glyceraldehyde‐3‐phosphate (GAPDH) (Chemicon, Billerica, MA, USA) and β‐actin (C4, Santa Cruz Biotechnology, Dallas, TX, USA). HPV58 E7 polyclonal antibody was custom synthesized from immunising rabbits with HPV58 E7 peptides (aa3‐20) using a 90‐day protocol with three boosters (ThermoFisher Scientific). Blots were visualized using ECL chemiluminescence (GE Healthcare) and images were captured using ChemiDoc™ Imagine System (Bio‐Rad, Hercules, CA, USA). Actin or GAPDH served as loading control for the blot. Band Intensities were quantitated by ImageLab and normalized with corresponding loading control level.
8
Co-Immunoprecipitation Assay Protocol
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HA (12CA5, Roche, Indianapolis, IN, USA) and Flag (F1804, Sigma, USA) antibodies were used in the co-immunoprecipitation assays. Total protein lysate was obtained in immunoprecipitation buffer. The total protein concentration of the supernatants was quantified using a Bio-Rad DC™ Protein Assay Reagent A/B/S (Bio-Rad, USA). 500 μg of total protein was mixed with 1 μg the primary antibody, or IgG, and the mixture were shaken on a rotating shaker at 4°C for 2 hours. Beads (Protein G Sepharose 4 Fast Flow, GE Healthcare Life Sciences, Piscataway, NJ, USA) were added to the mixture and shaken at 4°C for 1 hour. Then the beads were collected by centrifugation and washed three times by immunoprecipitation buffer. 2× sample loading buffer was added to the beads before boiling for 5 minutes. The supernatant was collected and used in the immunoblotting assays.
9
Protein Immunoblotting Analysis
10
Antibody Characterization for Protein Analysis
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Anti-Runx3 antibody (Ab40278) and anti-p53 antibody (Ab26) were obtained from Abcam (UK). Anti-phospho-p53 (#9284) was obtained from Cell Signaling Technology. Anti-Lamin antibody (#SC-20682) was obtained from Santa Cruz Biotechnology. Polyclonal anti-c-Myc-HRP (rabbit) (Sigma-Aldrich) and epitope-tag antibodies against Myc (9E10; Santa Cruz Biotechnology, Dallas, TX, USA), HA (12CA5; Roche Applied Science, Mannheim, Germany), and Flag (M2; Sigma, St. Louis, MO, USA) were purchased from the indicated vendors. Polyclonal anti-p27 and anti-p21 antibodies (rabbit) and monoclonal anti-cyclin D1 were purchased from Santa Cruz Biotechnology. Anti-phospho Erk (Thr-202, Tyr-204), anti-MEK1, and anti-phospho Akt (Ser-473) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-pRB was purchased from BD Bioscience (CA, USA). Tubulin antibodies were obtained from Lab Frontier (Seoul, Korea).
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