Cells were plated on coverslips and transfected following overnight incubation. Cells were fixed with 3% paraformaldehyde, permeabilized in 0.5% Triton-X100 for 10 min and blocked in 5% FBS diluted in PBS. Coverslips were incubated overnight with primary antibody HA (HA-7, H9658, Sigma-Aldrich) or FLAG (M2, F1804, Sigma-Aldrich), washed in PBS and incubated with anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, Life Technologies, Burlington, ON, Canada). Cells were mounted with ProLong Gold antifade with DAPI (Invitrogen). Visualization was done using an Olympus BX51 microscope with a 40x objective and images were captured with the Image-Pro Plus software (Media Cybernetics Inc., Bethesda, MD, USA). For quantification analysis, images were blinded by a third party.
Flag m2 f1804
Manufactured by Merck Group
Sourced in Canada, Germany, United States
The FLAG M2 (F1804) is a laboratory equipment product manufactured by Merck Group. It is designed for the detection and purification of proteins fused to the FLAG epitope tag. The product functions as an affinity matrix that can be used for immunoaffinity chromatography applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
β actin, by Merck Group (2 mentions)
P63 d2k8x, by Cell Signaling Technology (2 mentions)
Hklf4 af3640, by R&D Systems (2 mentions)
Krt14 ab7800, by Abcam (2 mentions)
Ha ha 7 h9658, by Merck Group (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Ab181547, by Abcam (1 mentions)
Eclipse 80i fluorescent microscope, by Nikon (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
10r g109a, by Biosynth (1 mentions)
Ho 1 adi osa 110, by Enzo Life Sciences (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ha 3f10, by Roche (1 mentions)
Alexa fluor 568 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
18 protocols using flag m2 f1804
1
Immunofluorescence Staining of Tagged Proteins
2
Antibody Characterization for Transcription Factors
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The anti-USF1 antibody is a rabbit polyclonal IgG directed against the C-terminus of USF1 (c-20, sc-229) or a rabbit polyclonal IgG directed against the amino acids 75–160 of USF1 (H-86, sc-8983), whereas the anti-USF2 antibody is a rabbit polyclonal IgG directed against the C-terminus of USF2 (c-20, sc-862). HA-tagged dominant negative USF mutant was detected with the HA specific mouse monoclonal antibody 12CA5 from Roche Diagnostics (Mannheim, Germany). PDX-1 was identified with a polyclonal antiserum generated by immunizing rabbits with recombinant mouse PDX-120 (link),61 . The mouse monoclonal antibody against CK2β (E-9, sc-46666) was purchased from Santa Cruz (Biotechnology Inc., Heidelberg, Germany). Detection of CK2α was performed by using the mouse monoclonal antibody 1 A562 (link). The mouse monoclonal antibody FLAG M2 (F1804), the β-actin and the α-tubulin (clone DM1A) antibody were from Sigma-Aldrich (Munich, Germany). Rabbit polyclonal serum #36 against nucleolin was prepared in our laboratory63 (link). Insulin was detected by the monoclonal rabbit antibody AB181547 from abcam (Cambridge, UK).
3
Immunofluorescence Staining of Transdifferentiated Fibroblasts
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Transdifferentiated and control fibroblasts grown on cover glasses were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. Cells were then washed twice with PBS and permeabilized with 2.5% Triton X-100 in PBS for 10 min. After two washes, cells were blocked in 10% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Cells were then incubated at 4°C overnight with primary antibodies in 5% BSA in PBS with 0.1% Tween 20 (PBST). Primary antibodies used were as follows: Flag (M2, F1804, Sigma-Aldrich), hKLF4 (AF3640, R&D Systems), p63 (D2K8X, Cell Signaling Technology), and KRT14 (ab7800, Abcam). The next day, cells were washed 4 × 10 min with PBST, followed by incubation with Alexa Fluor–conjugated secondary antibodies (Life Technologies) in 5% BSA in tris-buffered saline with Tween 20 for 45 min at 37°C. Cells were then washed 3 × 10 min in PBS, incubated with 4′,6-diamidino-2-phenylindole (0.5 μg/ml) in PBS for 5 min, and washed twice with PBS. The slides were then mounted using VECTASHIELD antifade mounting medium (H-1000, Vector Laboratories) and incubated overnight in the dark at room temperature. The slides were then imaged with a Nikon Eclipse 80i fluorescent microscope.
4
Antibody Characterization for Sperm Research
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SPINK2 rabbit polyclonal antibody was from Sigma‐Aldrich (HPA026813) and used at 1/1,000 for Western blot analysis. Sperm protein Sp56 and Golgi matrix protein GM130 (610822) mouse monoclonal antibodies were from QED Bioscience Inc. (used at 1/800 and 1/200, respectively). Promyelocytic leukemia zinc finger protein PLZF rabbit polyclonal antibody (Sc‐22839) was from Santa Cruz Biotechnology. Dpy19l2 antibodies were produced in rabbit as polyclonal antibodies raised against RSKLREGSSDRPQSSC and CTGQARRRWSAATMEP peptides corresponding to amino acids 6–21 and 21–36 of the N‐terminus of mouse Dpy19l2 (Pierre et al, 2012 ). DDK antibody was from OriGene (TA50011) or Sigma‐Aldrich (FLAG® M2F1804) and used at 1/10,000 for Western blot analysis. Acrosin antibody was previously described (Gallo et al, 1991 ) and is a gift from Denise Escalier.
5
Protein Extraction and Western Blotting
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Cells were lysed in radioimmunoprecipitation assay buffer containing 1% NP-40, 150 mM NaCl, 50 mM tris-Cl (pH 8.0), and 1% SDS supplemented with cOmplete, EDTA-free protease inhibitor (11873580001, Roche). Protein concentration was determined by bicinchoninic acid assay (BCA) protein assay (#23227, Life Technologies), after which equal amounts of proteins were loaded and separated by polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes. Antibodies used in this study were as follows: Flag (M2, F1804, Sigma-Aldrich), hKLF4 (AF3640, R&D Systems), p63 (D2K8X, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (10R-G109a, Fitzgerald), and KRT14 (ab7800, Abcam).
6
Protein Expression Analysis via Western Blot
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Western blot analyses were performed using 25 ~ 50 μg of cleared cell lysates as described previously28 (link). Antibodies used in this study were purchased from the following sources: phospho-AKT (S473) (#9271), AKT (#9272), phospho-ERK1/2 (T202/Y204) (#4370), and HER2 (#2247) from Cell Signaling Technologies, Inc. (Danvers, MA); HO-1 (ADI-OSA-110) from Enzo Life Sciences (Farmingdale, NY); β-actin, ERK1 (sc-94), NRF2 (sc-13032), and MRP5 (sc-5770) from Santa Cruz Biotechnology (Santa Cruz, CA); FLAG M2 (F1804) from Sigma; and GFP (ab290-50) from Abcam (Cambridge, MA). Chemiluminescence reagent was purchased from Santa Cruz Biotechnology or Thermo Scientific (Rockford, IL). Immunoprecipitation was performed as described previously16 (link).
7
Antibodies and Imaging for Xenopus Cardiac Myofibrils
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The peptide sequence used to raise a polyclonal rabbit antibody against Adprhl1 was: 248DNYDAEERDKTYKKWSSE265 (Cambridge Research Biochemicals). The sequence derives from mouse ADPRHL1, yet the antibody is also active against the Xenopus and human orthologs (aa residues 249-266). Other antibodies used were: Myosin A4.1025 (DSHB), Actin 20-33 A5060 (Sigma), Sarcomeric α-actinin EA-53 (abcam), FLAG M2 F1804 (Sigma), HA 3F10 (Roche), NKX2-5H-114 (Santa Cruz). Fluorescent dye-conjugated secondary antibodies (Jackson ImmunoResearch) and Alexa Fluor568-conjugated phalloidin (Invitrogen) were used to visualize myofibril components. Immunocytochemistry was performed on whole tadpoles and subsequently the hearts were dissected, mounted in 12 μl CyGEL Sustain (biostatus) and viewed using a Zeiss LSM5-Pascal confocal or a Zeiss Axioimager microscope. The confocal images of whole hearts captured 2 µm deep optical sections. Images of myofibrils were 1 µm optical sections, at a depth 1–2 µm below the outer (apical) myocardial surface. Cell number counts of the heart surface at stage 33 utilised cortical filament staining to discriminate cell boundaries while stage 37 and 40 ventricles used cell nuclei counts with Image J.
8
Immunoblotting Antibodies for Protein Analysis
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Immunoblotting was performed62 (link) using antibodies against: ATF4 (11815), phospho-eIF2 (3398), total eIF2 (9722), and Casp3 (9661) all from Cell Signaling Technology (Danvers, MA, USA); Flag-M2 (F1804) from Sigma-Aldrich (St Louis, MO, USA); Nupr1 (ab6028) from Abcam (Cambridge, MA, USA); Bex2 (sc48966), ATF3 (sc188), GADD34 (sc825), CHOP (sc7351), and ERK2 (sc154) were all from Santa Cruz Biotechnology (Dallas, TX, USA).
9
Western Blotting with Immunostained Proteins
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Western blotting with Immunostar® LD chemiluminescent substrates (Wako, 290–69904) was performed. Signals were detected with an LAS-3000 image analyzer (Fujifilm, Tokyo, Japan) and analyzed using Multigauge software (Fujifilm) as described previously.20,55,56 (link) LC3 (PM036), SQSTM1 (PM045), ATG5 (M153–3), BECN1 (PD017) and LMNB1/Lamin B1 (PM064) were purchased from MBL. LAMP2 (ab13524), CTSB (ab58802), CTSL (ab133641), TFEB (ab2636) and CIDEC (ab16760) were purchased from Abcam. CDKN1A (sc-397) and CASP1 (sc-514) were purchased from Santa Cruz Biotechnology. FLAG M2 (F1804) and ACTB/β-actin (A1978) were purchased from Sigma-Aldrich. TP53 (OP03) was purchased from Millipore. GAPDH (010–25521) was purchased from WAKO.
10
Immunostaining and Immunoblot Antibodies
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Mouse monoclonal antibodies against insulin (clone K36AC10, I2018, for immunostaining of paraffin sections), FLAG (M2, F1804), β-tubulin (DM1A, T9026), and glucagon (K79bB10, G2654) were from Sigma-Aldrich. Rabbit polyclonal antibodies against glucagon (ab92517), NNAT (ab27266), VAPB (ab72470), and ribophorin 1 (ab198508) and mouse monoclonal antibodies against PDI (RL90, ab2792) were from Abcam. Mouse anti-insulin (L6B10, 8138, for immunoblotting/immunofluorescence), anti–c-Myc (9B11, 2276), and anti-IRE1α (14C10, 3294) were purchased from Cell Signaling. MG132 was purchased from Sigma-Aldrich.
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