After protein samples were denatured at 70°C for 10 min under reducing conditions, proteins were resolved with 4–12% gradient 1D-SDS-PAGE (Invitrogen, Carlsbad, CA), and transferred to 0.45 µm nitrocellulose (or PVDF) membranes (Bio-Rad, Hercules, CA) using a Bio-Rad semi-dry transblotter. The membranes were blocked in LiCor blocking buffer (LiCor, Lincoln, NE) and then incubated overnight at 4°C in LiCor blocking buffer with 0.1% Tween 20 and the primary antibodies listed in Table 2 . All antibodies were individually optimized to determine ideal conditions within the linear range of detection for each assay, and that the primary antibody was present in excess. After antibody incubations, the membranes were rinsed in phosphate-buffered saline with 0.1% Tween (PBST) and probed with IR-dye labeled secondary antibodies (Li-Cor; 1:10,000) for 1 h at RT. The membranes were rinsed again with PBST and then with deionized water. Immunoblots were scanned using a LiCor Odyssey near-infrared scanner using the Odyssey V3.0.16 software package.
Semi dry transblotter
Manufactured by Bio-Rad
Sourced in United States
The Semi-dry transblotter is a laboratory instrument designed for the transfer of proteins from polyacrylamide gels to membranes, such as nitrocellulose or PVDF. It utilizes a semi-dry electroblotting method to facilitate the efficient transfer of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Blocking buffer, by LI COR (4 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (2 mentions)
Mini gel tank, by Thermo Fisher Scientific (2 mentions)
Odyssey scanner, by LI COR (2 mentions)
Odyssey 3.0 analytical software, by LI COR (2 mentions)
Irdye labeled secondary antibody, by LI COR (1 mentions)
Odyssey system, by LI COR (1 mentions)
Ab1801, by Abcam (1 mentions)
Ab11433, by Abcam (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Pdvf membrane, by Bio-Rad (1 mentions)
Hif 1α, by Thermo Fisher Scientific (1 mentions)
Hif 1α, by Merck Group (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Supersignal west femto kit, by Thermo Fisher Scientific (1 mentions)
Tris glycine sds page gel, by Bio-Rad (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Penta his hrp conjugate, by Qiagen (1 mentions)
11 protocols using semi dry transblotter
1
Western Blot Analysis of Protein Samples
2
Quantifying Brain-Derived Neurotrophic Factor
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To quantify BDNF levels, protein extracts (10 μg) were separated on 4–12% Nupage BisTris gels (Invitrogen, Carlsbad, CA). The proteins were transferred onto a Polyvinylidene fluoride membrane using a semidry transblotter (Bio-Rad). Blots were then probed with a BDNF antibody (1:1000, Cat. No. sc-546, Santa Cruz Biotechnology Inc., Dallas, Texas, USA). Secondary goat anti-rabbit 800CW antibody was used for protein detection (Licor Odyssey System, LiCor, Lincoln, NE). Quantifications were normalized to actin (1:1000, Cat. No. ab1801, Abcam, Cambridge, UK) and valosin-containing protein (1:5000, #ab11433, Abcam).
3
Western Blot Analysis of Brain Proteins
4
Western Blot Analysis Protocol
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Reducing buffer (6X: 170 mM Tris (pH 6.8), 4.5% SDS, 36% glycerol, 0.0018% bromephenol blue, 2% βME) was added to each sample, followed by a 10 min incubation at 70 °C. 10 µg of each sample was then loaded into Bolt™ 4–12% Bis-Tris Plus Gels (Invitrogen, Carlsbad, CA, USA), with fraction-matched ABC control tissue loaded on every gel. Gels underwent electrophoresis at 150 V for ~1 h, or until loading dye ran off the gel, using a Mini Gel Tank (Invitrogen, Carlsbad, CA, USA), then transferred to nitrocellulose membrane using a BioRad semi-dry transblotter (Hercules, CA). After transfer, blots were incubated in Ponceau (5% Acetic Acid, 0.1% Ponceau) for 5 min, followed by two one minute washes with tris-buffered saline (TBS) and an image was digitally acquired. Membranes were then incubated with TBS containing either 50% Li-Cor blocking buffer or 5% BSA for 1 h at RT. After blocking, blots were incubated overnight at 4°C in primary antisera diluted in TBS containing 0.1% Tween-20 (TBST) with either 50% Li-Cor buffer or 5% BSA (Supplemental Table 2 ). Blots were then washed with 1X TBST, incubated with secondary antibody, and washed again before being scanned on an Odyssey Infrared Imaging System (Li-Cor Biosciences) at a resolution of 169 µm and intensity level of 5.
5
Quantitative Analysis of Placental Hif1α
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Whole placental samples were sonicated and prepared as previously described [33 (link)]. 2 mM deferoxamine mesylate salt (DFO, Sigma-Aldrich, St. Louis, MO) was then added to prevent Hif1α degradation [34 (link)]. 100 μg of sonicate was boiled for 10 min in protein loading buffer and separated using 12% SDS-PAGE gel electrophoresis (BioRad, Hercules, CA). Proteins were transferred to a PDVF membrane (Bio-Rad) using a semidry transblotter (Bio-Rad). The membrane was probed with polyclonal antibodies raised against Hif1α (Invitrogen, Carlsbad, CA) and Actb (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were detected using a SuperSignal™ West Pico PLUS chemiluminescent substrate (Thermofisher, Waltham, MA) and band intensities were quantitated using Image Studio Lite software (LI-COR Biosciences, Lincoln, NE). Coomassie blue staining was performed to confirm equal loading of samples.
6
Western Blot Analysis of Purified rCNFy
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Samples containing 10 μg of protein from whole cells or MVs from Yersinia pseudotuberculosis YPIII pET31b-CNFy were separated on 4–20% Tris-glycine SDS-PAGE gels (Biorad, USA). Samples of purified rCNFy (0.2 µg, 0.1 µg and 0.05 µg) was also run on the same gel. Subsequently, the proteins were electro-blotted onto a polyvinylidene difluoride (PVDF) membrane (GE health care, USA) using a semi-dry transblotter (Biorad, USA). The membrane was then incubated for 2 h in a blocking solution (5% wt/vol skim milk (Difco, USA) in Tris-buffered saline (TBS; 20 mM Tris-HCl buffer (pH 7.6) containing 137 mM NaCl). After three sequential washes with TBS, the membrane was probed with a Penta·His-HRP conjugate (Qiagen, USA) for 2 h using 5% skim milk in TBS. The blot was then washed three times with TBST (TBS containing 0.05% (vol/vol) Tween-20). The signals were then developed using the Supersignal West Femto kit (Thermo scientific, USA) according to the manufacturer’s suggested protocol, and captured using the LAS 200 System (GE Healthcare, USA).
7
Western Blot Analysis Protocol
8
Immunodetection of Enzyme Proteins
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Crude extract protein was separated by sodium dodecyl sulfate (SDS) polyacrylamide-gel (10%) electrophoresis (PAGE) as previously described (Laemmli 1970). The gels were directly electroblotted onto Immobilon-P transfer membrane (Thermo Scientific, Rockford, IL, USA) using a semi-dry trans-blotter (Bio-Rad, Irvine, CA, USA) for immunodetection with the polyclonal antibodies against SPS (Sawitri et al. 2016 (link)). Proteins reacted with the antibodies were visualized with a secondary antibody of goat anti-rabbit IgG Alkaline phosphatase conjugate (Bio-Rad, Irvine, CA, USA) using the NBT/BCIP for color development (Bio-Rad, Irvine, CA, USA).
9
Quantitative Western Blot Analysis
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Western blot analysis was performed using protein extracted from cell lysates or supernatant culture medium, which were dissolved in loading buffer (5× solution of 50% glycerol, 10% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.5% bromophenol blue, and 250 mM Tris-HCl pH 6.8) and denatured for 5 min at 100 °C prior to electrophoresis. Proteins were analyzed using an 8–10% polyacrylamide gel and mid-range protein ladder (Beijing Cowin Biotech). Proteins were transferred for 90 min to polyvinylidene difluoride membranes using Bio-Rad semi-dry transblotters (Hercules, CA) and electroblotting (300 mA). Membranes were blocked for 1 hour at room temperature with Bovine Serum Albumin Blocking Buffer (Beijing Cowin Biotech). Strips were subjected to rabbit anti-sclerostin (1:1000; ab63097, Abcam, Cambridge, UK), rabbit anti-GAPDH (1:1000; EPR6256, ab128915, Abcam) antibodies over night at 4 °C. After incubation with polyclonal goat anti-rabbit IgG H&L Alexa Fluor® 790 (1:10000; Abcam), specific reactions were revealed with LI-COR’s Odyssey Infrared Imaging System and quantified by Odyssey 3.0 analytical Image Studio software (LI-COR Biotechnology, Lincoln, NE).
10
Western Blot Protein Detection Protocol
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Cell lysates were prepared by disrupting cell pellets in the Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) supplemented with 5% beta-mercaptoethanol (Sigma-Aldrich), and heated at 96°C for 5 min. Samples were then subjected in duplicate to 4–12% gradient SDS-PAGE (Invitrogen) and transferred onto PVDF membranes using BioRad semi-dry transblotters. Membranes were blocked with LiCor blocking buffer (LiCor, Lincoln, NE) for 1 h at room temperature, and probed with primary antibodies (described in Supplementary Table 1 ) diluted in LiCor blocking buffer containing 0.1% Tween. The membranes were washed twice for 10 min each in PBS with 0.1% Tween (PBST) and then probed with goat anti-mouse or goat anti-rabbit IR-Dye 670- or 800 cw-labeled secondary antisera diluted in LiCor blocking buffer containing 0.1% Tween and 0.01% SDS for 1 h at room temperature. The probed membranes were washed twice for 10 min in TBST, placed in water, and imaged using a LiCor Odyssey scanner. Boxes were manually placed around each band of interest, and near-infrared fluorescent values for raw intensity, with intra-lane background subtracted, were obtained using Odyssey 3.0 analytical software (LiCor).
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