[3H]-TPP (specific activity: >1.4 Ci/mmol; radiochemical purity: >98.2%) and [3H]-thiamin (specific activity: >12.8 Ci/mmol; radiochemical purity: >93.3%) were purchased from Moravek, Inc. Human-specific anti-TPPT (SLC44A4) affinity-purified rabbit polyclonal antibody was generated for us by Thermo Fisher Scientific; anti-THTR-1 (catalog no.: ab229680) rabbit polyclonal antibody was from Abcam; anti-THTR-2 (catalog no.: 13407-1-AP) rabbit polyclonal antibody was from Proteintech; anti-HIF-1α (catalog no.: 14179S) rabbit monoclonal antibody was from Cell Signaling Technology; anti-HIF-2α (catalog no.: NB100-122) rabbit polyclonal antibody was from Novus Biologicals; and anti-GAPDH mouse monoclonal antibody (catalog no.: sc-47724) was from Santa Cruz. The secondary antibodies, anti-rabbit IRDye-800 (catalog no.: 926-32211) and antimouse IRDye-680 (catalog no.: 926-68020), were purchased from LI-COR Bioscience. All other chemicals and reagents used in this studies were of analytical/molecular biology grade and purchased from established sources.
Anti mouse irdye 680
Manufactured by LI COR
Sourced in United States
The Anti-mouse IRDye-680 is a fluorescent dye conjugated to an antibody that specifically binds to mouse proteins. It is designed for use in Western blotting, immunohistochemistry, and other protein detection applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit irdye 800, by LI COR (17 mentions)
Odyssey blocking buffer, by LI COR (6 mentions)
Anti rabbit irdye 800cw, by LI COR (3 mentions)
Anti β actin sc 47778, by Santa Cruz Biotechnology (3 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Anti actin, by Merck Group (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Bullet blender high throughput homogenizer, by Next Advance (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Ncm460 cells, by INCELL (2 mentions)
Anti mouse irdye 800, by LI COR (2 mentions)
Sh sy5y, by American Type Culture Collection (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose filters 0.45 mm, by Merck Group (2 mentions)
Anti thtr 1 ab123246, by Abcam (2 mentions)
Anti sp 1 07645, by Merck Group (2 mentions)
Odyssey clx imager, by LI COR (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Odyssey nitrocellulose membrane, by LI COR (2 mentions)
25 protocols using anti mouse irdye 680
1
Radiolabelled Thiamin Uptake Assay
2
Protein Extraction and Western Blotting
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Cells/ mice tissues were lysed using lysis buffer (Cell Signalling) supplemented with a complete protease inhibitor cocktail (Roche Applied Science) and phenylmethylsulfonyl fluoride (Sigma-Aldrich Corp.). Tissues were further homogenized using 0.5 mm glass beads in a Bullet Blender high-throughput homogenizer (Next Advance, Inc.). After pelleting cellular debris by spinning at 16000 rpm at 4°C for 15 min, protein was quantified by Bradford assay. Detailed procedure for western blotting has been described previously [26 (link)]. In brief, 40–50 μg protein was added per lane of 4–12% Bis-Tris gels (Invitrogen Corp.). Primary antibodies were diluted in Odyssey blocking buffer (LI-COR Biosciences). Antibodies used included: anti-FXN (provided by Franco Taroni M.D., Istituto Besta) and anti-actin (#A2668, Sigma). Direct conjugated secondary antibodies (anti-rabbit IRdye800Cw and anti-mouse IRdye680 from LI-COR) were used to detect and quantify the signal of primary antibodies and imaged using a LI-COR Odyssey.
3
Western Blot Analysis of HER2 and OVA
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Tissue culture cells were harvested and lysed using RIPA buffer. Total lysate (20 µg) was ran on a gel using the Biorad Mini-PROTEAN system, followed by transfer using the Biorad semi dry transfer system. Primary antibodies used were rabbit anti-HER2 (1:1000, Cell Signaling), mouse anti-OVA (1:1000, Abcam) or rabbit anti-GAPDH (1:1000, Cell Signaling). Antirabbit IRDye 800 (1:1000, LICOR) and antimouse IRDye 680 (1:1000, LICOR) were used as secondaries. Blocking, probing, visualization and quantification were performed according to LI-COR Odyssey system specifications.
4
Affinity Pulldown of hELP Proteins
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First, 1 μM hELP1-3xFLAG or hELP1-3xFLAG/hELP3
was combined with 1 μM 10xHis-hELP2 in 200 μL reactions
and incubated with 50 mL of M2-FLAG resin for 1 h at 4 °C under
rotation. The M2-FLAG resin was isolated by centrifugation for 2 min
at 5000 × g, and the supernatant was collected
to analyze unbound protein. The resin was then washed three times
with 500 μL of pulldown buffer by resuspending the slurry in
buffer and collecting the resin by centrifugation. Bound proteins
were the eluted by incubation with 200 μL of 0.5 mg/mL 3xFLAG
peptide for 30 min at 4 °C (“Pulldown”). Then,
1 μM hELP1, hELP1/3, or hELP2 alone (“Input”),
along with FLAG elution fractions from each pulldown were loaded evenly
across 3 SDS-PAGE gels. Two gels were then transferred to nitrocellulose
membranes by wet transfer for 1.5 h at 100 V, and the third gel was
stained using Coomassie blue. Membranes were blocked for 1 h at RT
with a 5% skimmed milk solution then incubated with a 1:2000 dilution
of mouse anti-FLAG antibody (Sigma) or mouse anti-His antibody for
1 h at RT. Blots were then washed and incubated with a 1:10,000 dilution
of anti-mouse IRDye680 (Li-Cor BioSciences) secondary antibody. Blots
and stained gels were then imaged using a ChemiDoc MP.
was combined with 1 μM 10xHis-hELP2 in 200 μL reactions
and incubated with 50 mL of M2-FLAG resin for 1 h at 4 °C under
rotation. The M2-FLAG resin was isolated by centrifugation for 2 min
at 5000 × g, and the supernatant was collected
to analyze unbound protein. The resin was then washed three times
with 500 μL of pulldown buffer by resuspending the slurry in
buffer and collecting the resin by centrifugation. Bound proteins
were the eluted by incubation with 200 μL of 0.5 mg/mL 3xFLAG
peptide for 30 min at 4 °C (“Pulldown”). Then,
1 μM hELP1, hELP1/3, or hELP2 alone (“Input”),
along with FLAG elution fractions from each pulldown were loaded evenly
across 3 SDS-PAGE gels. Two gels were then transferred to nitrocellulose
membranes by wet transfer for 1.5 h at 100 V, and the third gel was
stained using Coomassie blue. Membranes were blocked for 1 h at RT
with a 5% skimmed milk solution then incubated with a 1:2000 dilution
of mouse anti-FLAG antibody (Sigma) or mouse anti-His antibody for
1 h at RT. Blots were then washed and incubated with a 1:10,000 dilution
of anti-mouse IRDye680 (Li-Cor BioSciences) secondary antibody. Blots
and stained gels were then imaged using a ChemiDoc MP.
5
Antibody Characterization and Validation
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NCM460 cells were from INCELL (San Antonio, TX), and [3H]-TPP (specific activity 1.3 Ci/mmol; radiochemical purity 97%) was from Moravek Biochemicals (Brea, CA); qPCR primers were from Sigma Genosys (Woodlands, TX); Other chemicals/reagents were from commercial vendors and were of analytical/molecular biology grade. Human and mouse specific anti-SLC44A4 polyclonal antibodies were generated for us by Alpha Diagnostics International (San Antonio, TX) and by Thermofisher (Rockford, IL), respectively. Human anti-ELF3 (AV31639) antibody was from Sigma-Aldrich (Saint Louis, MO); anti-CREB-1 (#9197), anti-ERK1/2 (#9102S), anti-Phospho ERK1/2 (#9101S) and anti-Phospho NF-κB (#3033S) were from Cell Signaling Technology (Danvers, MA); anti-NF-κB (ab16502) antibody from Abcam (Cambridge, MA); anti-β-actin (sc-47778) and anti-Lamin B (sc-6216) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibodies, anti-rabbit IRDye-800 and anti-mouse IRDye-680, were purchased from LI-COR Bioscience (Lincoln, NE).
6
Mitochondrial Protein Isolation and Western Blot Analysis
7
Western Blot Analysis of Oxidative Stress Markers
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Cells were lysed using lysis buffer (Promega Corp.) supplemented with a complete protease inhibitor cocktail (Roche Applied Science) and phenylmethylsulfonyl fluoride (Sigma-Aldrich Corp.). Tissues were further homogenized using 0.5 mm glass beads in a Bullet Blender high-throughput homogenizer (Next Advance, Inc.). After pelleting cellular debris by spinning at 16 000 rpm at 4°C for 15 min, protein was quantified by Bradford assay (66 ). For western blotting, 40–50 µg protein was added per lane of 4–12% Bis-Tris gels (Invitrogen Corp.). Primary antibodies were diluted in Odyssey blocking buffer (LI-COR Biosciences). Antibodies used included: anti-FXN (provided by Franco Taroni M.D., Istituto Besta), anti-HO1 (# sc-10789, Santa Cruz Biotechnology, Inc.), anti-NQO1 (#3187, Cell Signaling Technologies), anti-Gpx4 (#ab125066, AbCam), anti-nrf2 (sc-25820, Santa Cruz) and anti-actin (#A2668, Sigma). Direct conjugated secondary antibodies (anti-rabbit IRdye800Cw and anti-mouse IRdye680 from LI-COR) were used to detect and quantify the signal of primary antibodies and imaged using a LI-COR Odyssey.
8
Human Intestinal Epithelial Cell Study
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Human-derived intestinal epithelial NCM460 cells were obtained from INCELL (San Antonio, TX). [3H]-RF (specific activity: 21.2 Ci/mmol, radiochemical purity: > 98%) was purchased from Moravek Biochemicals (Brea, CA). Anti-hRFVT-1 polyclonal antibodies obtained from Abnova (Walnut, CA). Anti-Sp1, anti-hRFVT-2, anti-hRFVT-3 and anti-β-actin monoclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit IRDye-800 and anti-mouse IRDye-680 antibodies were purchased from LI-COR Bioscience (Lincoln, NE). All cell culture supplies, transfection and molecular biology reagents were obtained from commercial vendors.
9
Estrogen Regulation of Muscle Cell Proteins
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Primary human muscle cell line that was derived from the quadriceps muscle biopsy of a 5‐day‐old female infant (Edom et al., 1994 ) was kindly provided by Profs Moyly and Buttler‐Browne (INSERM, Paris, France). Proliferating mononuclear myoblasts were differentiated for 5 days to form multinuclear myotubes in E2 free environment, before exposing them to 10 nm E2, 100 nm E2 or mock for 6 h. All experiments were carried out in triplicate. Semi‐quantitative immunoblotting analysis was carried out to quantitate the protein expression of CUL4A, MT‐CO2, LDHA, ALDOA, and PKM. TUBA was used for normalization. The blots were scanned and quantified using Odyssey CLX Infrared Imager of Li‐COR and manufacturer's software. Cell culture mediums, fetal bovine serum, and antibiotics were obtained from Life Technologies, Inc. (Carlsbad, CA, USA) while insulin from Sigma‐Aldrich. CUL4A, MT‐CO2, and LDHA antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), ALDOA and PKM from Abcam (Cambridge, UK) and TUBA from Sigma‐Aldrich. As a secondary antibodies, anti‐rabbit IR Dye 800 or anti‐mouse IR Dye 680 (LI‐COR Biosciences, Lincoln, NE, USA) was used.
10
Thiamin Uptake in Human Neuroblastoma Cells
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