Irdye800 conjugated anti rabbit igg

Manufactured by Rockland Immunochemicals

IRDye800-conjugated anti-rabbit IgG is a fluorescently-labeled secondary antibody used for detection and quantification purposes in various immunoassays and imaging applications. It binds to rabbit primary antibodies and emits near-infrared fluorescence upon excitation, enabling sensitive and specific detection of target proteins or analytes.

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Lab products found in correlation

Mouse monoclonal anti α tubulin antibody, by Merck Group (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Odyssey infrared scanner, by LI COR (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Anti kras, by Merck Group (1 mentions) Anti α tubulin, by Cell Signaling Technology (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Heat inactivated fbs, by Lonza (1 mentions) Anti phospho p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions) Image studio lite program, by LI COR (1 mentions) Odyssey near infrared imager, by LI COR (1 mentions) Odyssey imaging system, by LI COR (1 mentions) Anti phospho p38 mapk rabbit polyclonal antibody, by Cell Signaling Technology (1 mentions) Anti phospho creb, by Cell Signaling Technology (1 mentions) 5α dihydrotestosterone dht, by Merck Group (1 mentions) Mouse monoclonal anti human β tubulin, by Merck Group (1 mentions) Irdye 680 conjugated goat anti mouse igg, by LI COR (1 mentions) Alexa fluor 680 anti mouse igg, by Thermo Fisher Scientific (1 mentions) Protease and phosphatase inhibitor tablet, by Roche (1 mentions) Laemmli sample buffer, by Bio-Rad (1 mentions)

8 protocols using irdye800 conjugated anti rabbit igg

KRAS G12C Mutant H358 Cell Line Characterization

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Human non-small cell lung cancer KRAS G12C mutant cell line H358 were grown in RPMI1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (Lonza) and incubated in humidified 37°C/5% CO2 incubator. Cells were washed once with 1x phosphate buffered saline (PBS) and then lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease and phosphatase inhibitors (Roche). Protein concentrations were determined by using the Pierce BCA protein assay kit (Life Technologies). Equal amount of protein was resolved on SDS-PAGE, and was subsequently transferred onto the nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat milk in TBS-0.1%Tween-20 and was then incubated with primary antibodies overnight at 4°C with gentle rotating. After washing, the membrane was incubated with fluorophore-conjugated secondary antibodies for 1 hour at room temperature. The membrane was then washed and scanned with an Odyssey Infrared scanner (Li-Cor Biosciences). Primary antibodies included anti-KRAS (Sigma #SAB1404011), anti-Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling Technology # 9101S), and anti-alpha-Tubulin (Cell Signaling Technology # 3873S). Secondary antibodies used were IRDye700-conjugated anti-mouse IgG and IRDye800-conjugated anti-rabbit IgG (Rockland, Gilbertsville, PA).

Lu J., Harrison R.A., Li L., Zeng M., Gondi S., Scott D., Gray N.S., Engen J.R, & Westover K.D. (2017). KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry. Structure (London, England : 1993), 25(9), 1442-1448.e3.

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Quantitative Analysis of UGDH Oligomerization

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SDS-PAGE gels from the crosslinking procedure were either stained with Gel Code Blue or analyzed by western blot probed with anti-UGDH rabbit polyclonal antibody. For western blots, indicated fractions were transferred to PVDF membranes and blocked with Pierce Superblock reagent before incubation with anti-UGDH primary at a dilution of 1:1000. Membranes were washed and proteins detected by secondary incubation with IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, 1:5000). Images were captured in the 800-channel of the Odyssey Near Infrared Imager and analyzed using the Image Studio Lite program (LI-COR Biosciences). We quantified the fluorescence intensity of bands representing monomeric and oligomeric UGDH. By dividing the total of each oligomeric species by the total intensity of all species, we obtained a normalized percentage of crosslinking efficiency. Dynamic range of the instrument was determined by similar analysis of a standard curve consisting of 12 concentrations of purified wild-type UGDH spanning 0.1 ng to 10 μg. All values for western imaging were analyzed in the linear range of the standard curve by adjusting the instrument settings appropriately and loading equal volumes from each sample such that the highest protein concentration per fraction loaded was ≤10 μg.

Grady G., Thelen A., Albers J., Ju T., Guo J., Barycki J.J, & Simpson M.A. (2016). Inhibiting hexamer disassembly of human UDP-glucose dehydrogenase by photoactivated amino acid crosslinking. Biochemistry, 55(22), 3157-3164.

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Quantifying Signaling Pathway Activation

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Lysates were solubilized in Laemmli sample buffer. Equal amounts of total protein, determined by a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL) with BSA as the standard, were separated by SDS‐PAGE and electrotransfer of proteins from the gel to PVDF membranes. Blots were probed with anti‐p38 rabbit polyclonal MAPK (Cell Signaling, Hartsfordshire, U.K.), and anti‐phospho‐CREB (Cell Signaling) antibodies. Binding was detected with IRDye 800‐conjugated anti‐rabbit IgG (Rockland, Gilbertsville, PA) or IRDye 680‐conjugated anti‐mouse IgG (Invitrogen, Carlsbad, CA) secondary antibodies. All data are expressed as integrated intensity following infrared detection (Odyssey Imaging system; LI‐COR Biosciences, Lincoln, NE). For p38 MAPK signaling, membranes were then stripped (2% SDS (w/v) in 25 mmol/L glycine, pH 2.0) and reprobed with anti‐phospho‐p38 MAPK rabbit polyclonal antibody (Cell Signaling). As a loading control, blots were then reprobed with anti‐α‐tubulin mouse monoclonal antibody, which was not significantly different between groups (Sigma, Castle Hill, NSW, Australia).

Strobel N.A., Matsumoto A., Peake J.M., Marsh S.A., Peternelj T., Briskey D., Fassett R.G., Coombes J.S, & Wadley G.D. (2014). Altering the redox state of skeletal muscle by glutathione depletion increases the exercise‐activation of PGC‐1α. Physiological Reports, 2(12), e12224.

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Androgen Receptor Signaling Pathway Evaluation

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All reagents were obtained from Fisher Scientific (Pittsburgh, PA) except
where indicated. 5-α-dihydrotestosterone (DHT) and DHT-glucuronide were
from Sigma (St. Louis, MO). Charcoal stripped FBS was from Hyclone (Logan, UT).
Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA
(DakoCytomation, Glostrup, Denmark, 1:1500 dilution); mouse monoclonal
anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated
anti-rabbit IgG (Rockland, Gilberstville, PA, 1:5000); IRDye 680 conjugated goat
anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, 1:5000); rabbit polyclonal
anti-human UGT2B17 (1:100) and rabbit polyclonal anti-human HSD17B3 (GeneTex,
Irvine, CA, 1:500); mouse monoclonal anti-human AR (Santa Cruz Biotechnology,
Inc, Dallas, TX, 1:500); rabbit monoclonal anti-human Notch 1 (Cell Signaling
Technology, Danvers, MA, 1:1000); mouse monoclonal anti-human HSD3B1 (1:100) and
rabbit monoclonal anti-human ARv7 (Abcam, Cambridge, MA, 1:1000). Rabbit
polyclonal anti-human UXS1 was raised against purified recombinant human UXS1,
residues 85–420 (Pocono Rabbit Farms). The UXS1 expression construct was
a gift from Nicola Burgess-Brown: Addgene plasmid # 39162.

Zimmer B.M., Howell M.E., Wei Q., Ma L., Romsdahl T., Loughman E.G., Markham J.E., Seravalli J., Barycki J.J, & Simpson M.A. (2016). Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential. Hormones & Cancer, 7(4), 260-271.

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Western Blot Analysis of JPH2 Protein

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Tissue lysates were made from hearts flash frozen in liquid nitrogen by pulverization followed by sonication in Radio-Immunoprecipitation Assay (RIPA) lysis buffer containing 10% CHAPS, 20 mM NaF, 1 mM Na₃VO₄, and 1× protease and phosphatase inhibitor tablets (Roche Diagnostics, Indianapolis, IN) each dissolved in 1mL water. Cellular debris was cleared from lysate by centrifugation (16,000 g for 25 min at 4°C). 75 μg of total protein was diluted in 2× Laemmli sample buffer (Bio-Rad, Hercules, CA) containing 5% β-mercaptoethanol, heated to 70°C for 10 minutes and resolved on a 6–12% gradient sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis gel. Proteins were electrotransferred to polyvinylidene difluoride (PVDF).
Membranes were blocked in 5% milk tris-buffered saline (TBS) for 1 hour. Primary antibody was suspended in 5% milk TBS solution and incubated with membranes for 3 hours at room temperature or overnight at 4°C followed by incubation in a 1:10,000 dilution of fluorescent secondary antibody (Alexafluor 680 anti-mouse IgG, Invitrogen or IRDye800 conjugated anti-rabbit IgG, Rockland) for 60 min at room temperature. Primary antibodies consisted of a custom polyclonal rabbit anti-JPH2 antibody (1:1,000), and monoclonal mouse anti-GAPDH (1:10,000, MAB374, Millipore). Blots were imaged using a Licor Odyssey two channel IR fluorescence scanner.

Reynolds J.O., Quick A.P., Wang Q., Beavers D.L., Philippen L.E., Showell J., Barreto-Torres G., Theurauf D.J., Doroudgar S., Glembotski C.C, & Wehrens X.H. (2016). Junctophilin-2 gene therapy rescues heart failure by normalizing RyR2-mediated Ca2+ release. International journal of cardiology, 225, 371-380.

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Western Blot Analysis of Phosphorylated Akt

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Total lysates were solubilized in Laemmli sample buffer. Equal amounts of total protein (20 μg) were separated by SDS‐PAGE and electrotransfer of proteins from the gel to PVDF membranes. The mouse monoclonal antibody for phospho‐Akt Ser⁴⁷³ (pSer⁴⁷³ Akt) was from Cell Signaling Technology (Hertfordshire, England). Binding was detected with IRDye^™ 680‐conjugated anti‐mouse IgG (Molecular Probes, Eugene, OR) fluorescent antibody via infrared detection (Odyssey Imaging system, LI‐COR Biosciences, Lincoln, NE). Membranes were then reprobed with rabbit polyclonal Akt antibody (Cell Signaling). Binding was detected with IRDye^™ 800‐conjugated anti‐rabbit IgG (Rockland, Gilbertsville, PA). As a loading control, blots were reprobed with anti‐α‐tubulin mouse monoclonal antibody (Sigma). A three‐point standard curve (e.g., 10 μg, 20 μg, and 40 μg of total protein from one sample) was run on every membrane to confirm a 100% increase in protein loaded resulted in a 100% increase in signal intensity for all proteins of interest (Mollica et al. 2009).

Wadley G.D., Laker R.C., McConell G.K, & Wlodek M.E. (2016). Endurance training in early life results in long‐term programming of heart mass in rats. Physiological Reports, 4(4), e12720.

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Western Blot Analysis of TRIP6 Protein

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Protein was extracted from tissues and cells using RIPA buffer. After centrifugation at 12,000 rpm for 20 min, the supernatant was collected for measure of total protein concentration by DC-protein assay (Bio-Rad) to keep the same loads. Protein samples were electrophoretically separated on a 10% SDS-PAGE, and transferred to a PVDF membrane. After blocking with 5% non-fat dry milk in TBST buffer, the membrane was incubated with Rabbit polyclonal anti-human TRIP6 antibody (1:500, ab137478, Abcam, Cambridge, UK) at 4 °C overnight. The membranes were washed three times with TBST buffer for 5 min, and then incubated with secondary antibody, anti-rabbit IgG conjugated IRDye800 (1:5000, Rockland Gilbertsville, CA) at room temperature for 2 h. Finally, membranes were scanned with an Odyssey infrared imaging system (LI-COR, Lincoln, NE), and analyzed with PDQuest7.2.0 software (Bio-Rad).

Zhu L., Xu X., Tang Y, & Zhu X. (2019). TRIP6 functions as a potential oncogene and facilitated proliferation and metastasis of gastric cancer. Biologics : Targets & Therapy, 13, 101-110.

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Quantifying PLIN5 Protein Levels

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The lysates were extracted, and total protein was isolated by 10% SDS/PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked in 5% fat-free milk for 1 h at room temperature, and then incubated with the primary antibodies overnight at 4 °C: rabbit polyclonal anti-PLIN5 (Abcam; 1:1,000; cat. no. ab222811), and mouse monoclonal anti-β-actin (1:2,500). After washing three times with Tris-buffered saline (TBS) containing 0.1% Tween-20, the membrane was incubated secondary antibody, anti-rabbit IgG conjugated IRDye800 (1:5,000, Rockland Gilbertsville, CA) for 2 h at room temperature. At last, the protein bands were tested using the ECL Plus kit (ZSbio).

Zhao Y., Xu D., Wan Y, & Xi Q. (2020). Methylation of PLIN5 is a crucial biomarker and is involved in ovarian cancer development. Translational Cancer Research, 9(4), 2919-2930.

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