Whole cell protein extracts were prepared in RIPA lysis buffer (Sigma) containing a protease inhibitor cocktail (Roche) and PMSF 10 µg/mL, separated on a 10% SDS-PAGE gel (30 µg/lane), then transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Freiburg, Germany) by electro-blotting. Blots were blocked in 1:1 Odyssey buffer in PBS and developed in Odyssey buffer in 1x PBS/Tween 20. For detection of specific proteins, blots were incubated with the primary antibody against DAT/Slc6a3. β-Actin was used as a loading control. Secondary antibodies were conjugated with IRDye 680 or 800 (1:10,000; LI-COR Biosciences) and blots were analyzed on the Odyssey Infrared Imaging System (LI-COR Biosciences). The ratio of the respective protein band to the loading control was used for semi-quantitative analysis (Image Studio Light®, Odyssey).
Irdye 680 or 800
Manufactured by LI COR
Sourced in Germany
The IRDye 680 or 800 are near-infrared fluorescent dyes developed by LI-COR for use in a variety of applications, including in vitro and in vivo imaging, Western blotting, and ELISA. These dyes have specific excitation and emission wavelengths that make them suitable for detection and quantification of target molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (6 mentions)
Nitrocellulose membrane, by Cytiva (4 mentions)
Ripa lysis buffer, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Phosphosafe buffer, by Merck Group (3 mentions)
Ripa lysis buffer, by Cell Signaling Technology (3 mentions)
Odyssey clx, by LI COR (2 mentions)
Odyssey clx scanner, by LI COR (1 mentions)
Ab2413, by Abcam (1 mentions)
Ab178684, by Abcam (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Poly 1 c, by Merck Group (1 mentions)
Alpha tubulin, by Merck Group (1 mentions)
Ab36810, by Abcam (1 mentions)
Ab77256, by Abcam (1 mentions)
Ab10484, by Abcam (1 mentions)
Ab70250, by Abcam (1 mentions)
Ab18192, by Abcam (1 mentions)
Ab8898, by Abcam (1 mentions)
Ab6002, by Abcam (1 mentions)
14 protocols using irdye 680 or 800
1
Western Blot Analysis of DAT Protein
2
Western Blot Analysis of Protein Extracts
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Whole cell protein extracts were prepared in PhosphoSafe Buffer (Sigma Aldrich, Darmstadt, Germany) or RIPA lysis buffer (Sigma Aldrich) containing a protease inhibitor cocktail (Roche) and PMSF 10 µg/mL, separated on a 10% SDS-PAGE gel (30 µg/lane), transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, 79111 Freiburg, Germany) by wet-blotting or electro-blotting. Blots were blocked and developed in Odyssey buffer or 5% skim milk in 1× PBS/Tween 20. For detection of specific proteins, blots were incubated with primary antibodies (see Supplementary Table S1 ). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Secondary antibodies were conjugated with IRDye 680 or 800 (1:10,000; LI-COR Biosciences, 61352 Bad Homburg, Germany) and blots were analyzed on the Odyssey Infrared Imaging System (LI-COR Biosciences). The ratio of the respective protein band to the loading control was used for semiquantitative analysis (Image Studio Light®, Odyssey).
3
Immunoblot Analysis of Tissue and Cell Proteins
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Immunoblot analysis of proteins from tissue samples was done using urea homogenate as described below for MS analysis. Protein lysates from cell lines were prepared using a modified RIPA buffer as described above. Proteins were separated on a precast gradient (4–12%) Bis‐Tris gel and transferred to a nitrocellulose membrane. The membrane was blocked with Odyssey blocking buffer for 1 h at room temperature and incubated with primary antibodies against pAkt‐S473 (CST4060), total Akt (CST2920), AL1A1 (CST12035), CEAM1/2 (CST14771), INSR(CST3020), pINSR (CST3024), EGFR (CST4267), pERK (CST9101), F16P1 (CST59172), fibronectin (Abcam ab2413), GSTM1/2/4/5 (Abcam ab178684), and PYC (Abcam 128952) overnight at 4°C. GAPDH (sc‐20357) or β‐actin (CST4967) was used as loading control. The next day, the blot was incubated with secondary antibodies coupled to IRDye 680 or 800 (LI‐COR) for 1 h at room temperature, and immunocomplexes were visualized using an Odyssey scanner CLx (LI‐COR) and analyzed with Image Studio version 3.1 and 5.0.
4
Western Blot Analysis of Immune Cells
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Cultured BMDMs and DCs were harvested after 24 h of stimulation with vehicle, ovalbumin (EndoGrade, 50 µg/ml), LPS (0.1 µg/ml), or poly (I:C) (Sigma-Aldrich, 10 µg/ml). Whole cell protein extracts and tissue protein were prepared in radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling) containing a protease inhibitor cocktail (CompleteTM, Roche Diagnostics) and phenylmethylsulfonyl fluoride (PMSF, 10 µg/ml). The proteins were separated by 10–16% SDS-PAGE, transferred onto nitrocellulose membranes (Amersham Pharmacia) by electroblotting, incubated with the primary antibodies listed in Supplementary Table 2 and detected with secondary antibodies conjugated to IRDye 680 or 800 (1:10000; LI-COR Biosciences). Beta-actin or GAPDH were used as the loading control. Blocking was achieved with 5% skimmed milk in 0.1% Tween 20 in 1x PBS. All incubations were performed in Tris-buffered saline containing 0.5% Tween 20. The blots were visualized and analyzed on the Odyssey Infrared Imaging System (LI-COR Biosciences) and quantified with Image Studio Lite, and the ratio of each protein band versus the control band was used for quantitative assessment.
5
Western Blot Analysis of Chromatin Proteins
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MEFs were lysed in RIPA buffer with protease and phosphatase inhibitors and lysates were processed using standard methods. We used the following primary antibodies: VHL (sc-5575, 1:250; Santa Cruz), PBRM1 (A301-590A, 1:1000; Bethyl Laboratories), alpha-Tubulin (T5168, 1:2000; Sigma), RAD51 (sc-8349, 1:100; Santa Cruz), RPA32 (A300-244A, 1:500; Bethyl Laboratories), pATM (ab36810, 1:1000; Abcam), ATM (ab85213, 1:1000; Abcam), pDNA-PKc (ab18192, 1:1000; Abcam), DNA-PKc (ab70250, 1:1000; Abcam), pCHK1 (#2348 S, 1:500; Cell Signalling), CHK1 (#2360, 1:1000; Cell Signalling), H3K9me3 (ab8898, 1:1000; Abcam), H3K27me3 (ab6002, 1:1000; Abcam), H3K9me2 (ab8898, 1:1000; Abcam), H4K20me3 (ab9053, 1:1000; Abcam), HP1a (ab77256, 1:1000, Abcam), KAP1 (ab10484, 1:1000; Abcam) and H3 (4499, 1:2000; Cell Signalling). Secondary antibodies were conjugated to IRDye 680 or 800 (Li-Cor). Fluorescent signals were imaged using the Odyssey Infrared Imaging System (Li-Cor). Western blot band quantifications were performed with Fiji58 (link). Uncropped scans of presented blots are shown in Supplementary Fig. 7 .
6
Western Blot Quantification Protocol
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Cells were lysed in RIPA buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). Samples were separated by SDS–PAGE and transferred to cellulose membrane. Transferred membranes were probed with primary antibodies diluted in LI-COR (Lincoln, NE) blocking buffer (with 0.05% Tween-20). Secondary antibodies were rabbit or mouse IR-Dye 680 or 800 (LI-COR). Membranes were imaged using a LI-COR Odyssey scanner. Boxes were manually placed around each band of interest, which returned near-infrared fluorescence values of raw intensity, with intralane background subtracted using LI-COR Odyssey 3.0 analytical software. The fluorescence value for each protein of interest was normalized to the in-lane value of β-actin, and this normalized ratio was averaged from duplicate or triplicate lanes. Data were analyzed using the t test. Measures were considered significant when p < 0.05. Error bars are SEM.
7
Immunoblotting for Protein Expression
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The total protein lysate was collected by homogenization of flash frozen spleen, thymus, lymph nodes, skeletal muscle, or spinal cord in RIPA lysis buffer (Cell Signalling). Protein concentrations were determined using the Bradford assay (Bio-Rad). Protein extracts were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis and examined by immunoblot, as previously described (76 (link)) with modified blocking conditions where Odyssey blocking buffer (Li-Cor 927-40000) replaced 5% milk. Revert Total protein stain (Li-Cor 926-11010) was used as per the manufacturer’s protocol. Primary antibodies used were as follows: Anti-Smn (BD Transduction, 610647 - 1:2000), CD3 (DAKO #2018-11 - 1:1000), CD19 (Abcam, ab197895 - 1:1000) and alpha-tubulin (Abcam, ab4074 – 1:1000). Secondary antibodies used were IRDye 680 or 800 (Li-Cor - 1:10000 to 1:20000). Signals were detected with Odyssey CLx (Li-Cor). The results were normalized to total protein or tubulin.
8
Western Blot Analysis of Protein Samples
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OBSC sections (4-6) were pooled and lysed with Tris buffer and centrifuged at 21,000g to remove the nuclear fraction as we previously reported (48 (link)). Protein concentrations were measured using a Pierce BCA assay kit with the manufacturer’s instructions (ThermoFisher Scientific). Samples were diluted in RIPA and DTT containing reducing buffer (Pierce TM Cat. # 39000) to a final amount of 40 μg protein per well. Protein samples were separated on a 4–15% Ready Gel Tris-HCL gel (BioRad) and transferred onto PVDF membranes (BioRad). PVDF membranes were incubated at 4°C overnight with primary antibody. The following day, secondary antibody incubation (Li-Cor IRDye® 680 or 800) was performed, and membranes visualized using the LiCor Odyssey™ imaging system. Values for proteins of interest were normalized to beta-actin expression for each sample.
9
Quantitative Western Blot Analysis
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Whole cell protein extracts were prepared in RIPA lysis buffer (Cell Signalling) containing a protease inhibitor cocktail (Complete™; Roche Diagnostics, Mannheim, Germany) and PMSF 10 μg/mL. Tissue samples were homogenized in PhosphoSafe Buffer (Sigma) enriched with 10 μmol/L Pefabloc (serine‐protease inhibitor). Proteins were separated by 12% SDS‐PAGE, transferred to nitrocellulose membranes (Amersham Pharmacia) by wet‐blotting and detected using the anti‐human GCH1 (Sigma) and secondary antibodies conjugated with IRDye 680 or 800 (1:10 000; LI‐COR Biosciences, Bad Homburg, Germany). Beta‐actin was used as a loading control. Antibodies are listed in Table S1 . Blocking was achieved with 5% skimmed milk in 0.1 Tween 20 in 1× PBS. All incubations were done in Tris‐buffered saline containing 0.1% Tween 20 or in Odyssey buffer. Blots were visualized and analysed on the Odyssey Infrared Imaging System (LI‐COR Biosciences), quantified with Image Studio Lite (LICOR Biosciences) and the ratio of the respective protein band to the control band was used as semi‐quantitative readout.
10
Western Blot Protein Quantification
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Whole cell protein extracts were prepared in RIPA lysis buffer (Sigma) or PhosphoSafe Buffer (Sigma) containing a protease inhibitor cocktail (Roche) and PMSF 10 μg/ml, separated on a 12% SDS-PAGE minigel (Amersham; 50 μg/lane) and transferred to nitrocellulose membranes by electro-blotting. Blots were blocked and developed in Odyssey blocking buffer in 1xPBS/Tween 20. Blots were incubated with primary antibodies overnight (Suppl. Table 4 ), and β-actin was used as loading control. Secondary antibodies conjugated with IRDye 680 or 800 (1:1000; LI-COR Biosciences, Bad Homburg, Germany) were used for detection. Blots were sequentially developed and analyzed on the Odyssey Infrared Imaging System (LI-COR Biosciences), superimposed and quantified using ImageStudio Light.
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