After 2 weeks of injections, the CKO and control mice were tested to determine if the Cre recombinase induced activity had led to menin deletion. Hippocampus tissue was obtained for western blots to confirm the absence of menin in the CKO mice compared to the controls. Membranes were visualized using a LI-COR Odyssey infra-red imager and analyzed using the gel analysis tool in ImageJ.
Infrared imager
Manufactured by LI COR
Sourced in United States
The Infrared Imager is a device that captures and displays infrared images. It detects and measures infrared radiation, which is invisible to the human eye, and converts it into a visual representation. The core function of the Infrared Imager is to provide a non-invasive way to visualize and analyze infrared radiation patterns.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Sds page gel, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Odyssey, by LI COR (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Irdye 800cw streptavidin, by LI COR (1 mentions)
Sds page gel electrophoresis, by Bio-Rad (1 mentions)
0.22 m pvdf membrane, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Odyssey blocking solution, by LI COR (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
α tubulin tu 02, by Santa Cruz Biotechnology (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Mouse anti myc antibody, by Cell Signaling Technology (1 mentions)
Donkey anti rabbit 800 secondary antibody, by LI COR (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
13 protocols using infrared imager
1
Menin Deletion Confirmation in CKO Mice
2
Immunoprecipitation and Western Blot Analysis
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Myotubes were homogenized in RIPA buffer (see above). Dynabeads® Protein A were prepared according to the manufacturer's instructions (Invitrogen; 10001D). For pre‐clearing, equal amounts of protein were incubated with rabbit IgG‐conjugated Dynabeads® for 4 h at 4°C. The supernatant was incubated with primary antibody for overnight at 4°C, followed by incubation with Dynabeads® for 4 h at 4°C; the supernatant was discarded, and the beads were washed 3 times with RIPA buffer; the immunoprecipitant was eluted with SDS sample buffer, followed by Western analysis.
For Western blot analysis, cell lysates or whole muscle tissues were homogenized in RIPA buffer. Samples were centrifuged for 10 min at 18,000 × g at 4°C. Protein concentrations of the supernatants of the total lysates were measured using the Bio‐Rad Protein Assay (Bio‐Rad Laboratories, Inc.). Equal amounts of protein were run on SDS–PAGE gels (Invitrogen, Carlsbad, CA) followed by electro‐transfer onto nitrocellulose membranes (Invitrogen, Carlsbad, CA). Membranes were blocked in 1∶1 PBS and Odyssey Blocking Buffer (LI‐COR Biosciences, Lincoln, NE), incubated with primary antibodies overnight at 4°C, washed, incubated with secondary antibodies and washed again. Blots were scanned on an infrared imager (LI‐COR Biosciences).
For Western blot analysis, cell lysates or whole muscle tissues were homogenized in RIPA buffer. Samples were centrifuged for 10 min at 18,000 × g at 4°C. Protein concentrations of the supernatants of the total lysates were measured using the Bio‐Rad Protein Assay (Bio‐Rad Laboratories, Inc.). Equal amounts of protein were run on SDS–PAGE gels (Invitrogen, Carlsbad, CA) followed by electro‐transfer onto nitrocellulose membranes (Invitrogen, Carlsbad, CA). Membranes were blocked in 1∶1 PBS and Odyssey Blocking Buffer (LI‐COR Biosciences, Lincoln, NE), incubated with primary antibodies overnight at 4°C, washed, incubated with secondary antibodies and washed again. Blots were scanned on an infrared imager (LI‐COR Biosciences).
3
Quantitative detection of tagged proteins
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Protein content in cell culture lysates and eluates from biotin/streptavidin or acyl-RAC mediated protein purification was determined by the BCA assay (Pierce, Thermofisher Scientific) and mixed with 4x reducing Laemmli buffer to yield the indicated protein amounts in 1x Laemmli buffer. Unless stated otherwise, samples were boiled for 5 min. Proteins were then separated by SDS-PAGE gel electrophoresis (BioRad) and blotted onto PVDF membranes. Antibodies were prepared in blocking buffer (PBS-T in 5% BSA) as follows: mouse polyclonal anti-myc (serum from the lab); mouse monoclonal anti-HA (1:10 000; Thermo-Fisher #26183); mouse or rabbit monoclonal anti-FLAG (1:10 000; Thermo-Fisher #14-6681-82 and #701629). Quantitative immunodetection of tagged proteins and biotinylated proteins was carried out using a LI-COR infrared imager after incubation with the following antibodies/reagents: Goat anti-Mouse IgG (1:10 000; LI-COR #925–68020 IRDye 680LT Goat anti-Mouse IgG); Goat anti-Rabbit IgG (1:10 000; LI-COR # 926–68021 IRDye 680LT Goat anti-Mouse IgG), fluorescent Streptavidin 1:10 000; LI-COR IRDye® 800CW Streptavidin.
4
Western Blot Analysis of Skin Proteins
5
RANK Overexpression and Mutations in HeLa Cells
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HeLa cells were cultured in six-well plates at a density of 1×105 cells/ml, and after 24 h the cells were transfected with WT-RANKmyc, W434X-RANKmyc or G280X-RANKmyc using the JetPRIME transfection reagent. After 48 h, the cells were prepared in a radioimmunoprecipitation assay buffer (RIPA; deoxycholic acid (12.5 mM), SDS (3.5 mM) and IGEPAL (1%) in PBS) containing 1% protease inhibitor cocktail for western blot analysis as described previously (Crockett et al. 2011 (link)). The proteins on the PVDF membrane were probed with mouse anti-RANK antibody (Imgenex, clone 9A725, #IMG-128A) followed by donkey anti-rabbit-800 secondary antibody (LI-COR Biosciences, Cambridge, UK) and then with mouse anti-Myc antibody (Cell Signaling Technology, Danvers, MA, USA) followed by donkey anti-mouse-680 secondary antibody (LI-COR Biosciences). The blots were analysed on an LI-COR Infrared imager with Odyssey analysis software.
6
Protein Isolation and Western Blotting
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For total protein isolation, the white part of gastrocnemius muscle (n = 4–6 from each category) was homogenized in radioimmunoprecipitation assay (RIPA) buffer (PBS containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease/phosphatase inhibitor cocktail (#5872, Cell Signaling Technology). Total protein lysates were centrifuged for 15 min at 16,000 × g at 4°C. Proteins were separated by SDS-PAGE gels (Invitrogen, Carlsbad, CA, USA) by loading equal amount. Separated proteins were transferred onto nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA). Membranes were then treated with blocking buffer (5% nonfat milk), incubated with primary antibodies, washed and incubated with the appropriate secondary antibodies. Horseradish peroxidase (HRP)-chemiluminescence was developed using Azure Radiance plus kit and scanned on imager (Azure Biosystems). Alternatively, membranes were treated with Odyssey Blocking Buffer (LI-COR Biosciences) and incubated overnight with primary antibodies. After washing, membranes were incubated with fluorophore conjugated secondary antibodies and scanned on an infrared imager (LI-COR Biosciences).
7
Immunoblot Protein Expression Analysis
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Immunoblot was performed as described56 (link). Primary antibodies for pSTAT3-Y705 (9145 L) STAT3 (4904 S), pERK (4370 S), ERK (4695 S), PARP (9542 L), and β-actin (4967 S) were purchased from Cell Signaling Technology, Inc. Primary antibody for α-SMA was purchased from Abcam (ab5694). Secondary antibody was purchased from LI-COR (926–32211). Blots were imaged on a LI-COR infrared imager. Densitometry was performed using ImageJ software. All densitometric calculations represent an average of 3 biological replicates.
8
Immunoblotting Analysis of Cardiac Proteins
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Proteins were obtained from non-infarct LV homogenates or cultured cardiomyocytes as described above. Immunoblotting was performed by using antibodies against CEACAM1 (Santa Cruz Biotechnology, Dallas, USA), cytochrome C (Abcam, Cambridge, UK), cleaved caspase-3 (Abcam, Cambridge, UK), Bax (Santa Cruz Biotechnology), or COXIV(Cell Signaling Technology, Danvers, USA). After blocking the membrane with skim milk for 1 h, incubation with the primary antibodies was done overnight at 4 °C. The secondary antibody was Alexa Fluor 680 (Molecular Probes), in a 1:15000 dilution with NFM for 1 h at room temperature. Proteins were visualized with a LI-COR infrared imager and quantitative densitometric analysis was performed using Odyssey software (version 1.2, LI-COR).
9
Western Blot Analysis of Cell Lysates
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Cells were lysed in radioimmunoprecipitation assay buffer (RIPA) (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) supplemented with Complete Protease Inhibitor Cocktail Tablets (Roche, Basel, Switzerland) immediately prior to use. The antibodies used in this study were: β-actin (Sigma, 20-33), PTEN (Cell Signalling, 9552), p53 (Abcam, ab240) and DNMT1 (Cell Signalling, D63A6). Images were revealed using the LI-COR infra-red imager using LI-COR infra-red secondary antibodies.
10
Western Blot Protein Quantification and Analysis
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Cells were lysed with 100 uL lysis buffer (50mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM NaF, 10% Glycerol) containing Complete and PhosStop phosphatase inhibitors (Roche, Mississauga, ON, Canada). Whole cell lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. The protein concentration in cleared whole cell lysates was identified using BCA kit (Thermo Scientific, Rockville, IL, USA). Protein lysates (30–50 ug) were analyzed by SDS-PAGE (6–10%) and transferred by Western Blot to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked in PBS containing 5% BSA or TBS Odyssey buffer (Licor, Lincoln, NE, USA) if probing with phospho-antibodies. The primary antibody dilution was conducted as per the manufacturer’s protocol. Membranes were washed 3x with TBST followed by appropriate IRdye-conjugated secondary antibodies for 1h. Blots were washed again 3X for 5 min with TBST. Detection was via image analysis using a LICOR infrared imager and Image Studio Lite version 3.1 software.
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