Iblot gel transfer device

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The IBlot Gel Transfer Device is a laboratory equipment used for the transfer of proteins from polyacrylamide gels to membranes, a crucial step in western blotting analysis. The device uses an electric current to facilitate the efficient transfer of proteins, enabling researchers to study their expression and interactions.

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174 protocols using iblot gel transfer device

Western Blot Analysis of Sirt3 Protein

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Isolated neutrophils or washed platelets were homogenized in ice-cold RIPA buffer supplemented with protease inhibitor cocktail (Sigma). Proteins were resolved on a 4–20% SDS/PAGE gel and electroblotted onto PVDF membranes using an iBlot Gel Transfer Device (Thermo Fisher Scientific). The membranes were blocked with 5% BSA in TBS/0.05% Tween-20 for 2 hrs at room temperature and incubated overnight at 4°C with rabbit monoclonal anti-Sirt3 (Cell Signaling, #5490, 1:500). Membranes were then probed with a horseradish peroxidase–conjugated anti-rabbit IgG secondary antibody (Bio-Rad Laboratories, 1:4000). Detection was carried out with a Pierce ECL Western Blotting substrate (Thermo Fisher Scientific). Equal loading was confirmed by probing for anti-GAPDH mouse monoclonal antibody (Ambion, #AM4300, 1:20,000) or anti-β-Tubulin rabbit monoclonal antibody (Cell Signaling, #5346, 1:1000).

Hayashi H., Cherpokova D., Martinod K., Witsch T., Wong S.L., Gallant M., Cifuni S.M., Guarente L.P, & Wagner D.D. (2017). Sirt3 deficiency does not affect venous thrombosis or NETosis despite mild elevation of intracellular ROS in platelets and neutrophils in mice. PLoS ONE, 12(12), e0188341.

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Western Blot Protein Detection Protocol

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Sample proteins were extracted using RIPA buffer supplemented with protease inhibitor (Sigma, P8340) and kept frozen at −20°C until used. The samples were equalized using the BCA assay (23225, Thermo Fisher Scientific) and mixed with Laemmli buffer (1610747, Bio-Rad) supplemented with 5% β-mercaptoethanol and loaded in pre-cast gels (4561093, Bio-Rad) for PAGE-SDS electrophoresis. Proteins were transferred from the gel to a PVDF membrane using pre-assembled iBlot dry transfer stacks (IB401001, Thermo Fisher Scientific) in the iBlot gel transfer device (Thermo Fisher Scientific). After blocking with 5% non-fat milk in TBS with 0.5% Tween-20, membranes were incubated o/n at 4°C with the following primary antibodies: rabbit anti-PDGFRα (PA5-16571, Thermo Fisher Scientific, RRID:AB_10981626) and mouse anti-βactin (A5441, Sigma, AB_476744). Membranes were then washed and incubated with secondary antibodies: goat anti-rabbit IRDye 800 CW (925-68070, Li-Cor, AB_2651128) and goat anti-mouse IRDye 680RD (925-32221, Li-Cor, AB_621843), washed and let dry before being developed using an Odyssey Clx LI-COR Imaging system (LI-COR, NE, United States).

Velasco-Estevez M., Koch N., Klejbor I., Caratis F, & Rutkowska A. (2022). Mechanoreceptor Piezo1 Is Downregulated in Multiple Sclerosis Brain and Is Involved in the Maturation and Migration of Oligodendrocytes in vitro. Frontiers in Cellular Neuroscience, 16, 914985.

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Western Blotting of Transfected Cell Lysates

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One-fifth portion of the transfected cells was lysed using CytoBuster Protein Extraction Reagent (EMD Millipore). The clarified lysates were diluted 50-fold into CytoBuster and run on two 8% SDS-PAGE gels, with WT sample as a standard on each gel. To further standardize the blots, the gels were cut at the 70-kDa marker, so that the upper half contained the Hsp90 control band and the bottom half hTET2-CS. The Hsp90 halves of both gels were transferred together onto a single PVDF membrane, and the two TET halves were transferred onto another membrane, using an iBlot Gel Transfer Device (Thermo). Membranes were blocked for 2 h at room temperature with 5% (w/v) milk in Tris-buffered saline with 0.1% (v/v) Tween-20 (TBST), washed 3× with TBST, blotted with primary 1:10,000 anti-FLAG M2 (Sigma, cat. no. F1804) or 1:1,000 anti-Hsp90α/β (Santa Cruz Biotechnology, cat. no. sc-13119) antibodies at 4 °C overnight, washed, blotted with secondary 1:5,000 goat anti-mouse-HRP (Santa Cruz Biotechnology, cat. no. sc-2005) for 2 h, washed, and imaged with Immobilon Western Chemiluminescent HRP Substrate (Millipore) on a Fujifilm LAS-1000 imager with 30-s exposures.

Liu M.Y., Torabifard H., Crawford D.J., DeNizio J.E., Cao X.J., Garcia B.A., Cisneros G.A, & Kohli R.M. (2016). Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine. Nature chemical biology, 13(2), 181-187.

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Western Blot Analysis of MCF-7 Cells

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Traditional Western blots employed a previously published protocol with minor changes.^{15 (link)} Briefly, MCF-7 cells were treated with compounds for 24 hours before collection and sonication. Protein concentration was determined using BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher, Waltham, MA, USA), and 30 μg of protein was added to each well of a 10 % bis-tris gel with LDS sample buffer (Thermo Fisher, Waltham, MA, USA). Gel was transferred using the iBlot™ gel transfer device with PVDF membrane iBlot™ transfer stacks (Thermo Fisher, Waltham, MA, USA). Primary antibodies (1:1000) (ERα, PA1-308; CYP1A1, PA1-340; Thermo Fisher, Waltham, MA, USA) (β-actin, 4967S, Cell Signaling, Danvers, MA, USA) were added to blocked membranes and incubated at 4 °C overnight. Secondary antibody was added (1:1000) (Horseradish Peroxidase-linked anti-rabbit IgG, 7074S, Cell Signaling, Danvers, MA, USA) to membranes for 1 hour at room temperature. Then, SuperSignal™ West Femto (Thermo Fisher, Waltham, MA, USA) was added and imaged on a FluorChem™ system (Protein Simple, Santa Clara, CA, USA). Quantification used ImageJ software to determine ratios of protein of interest over β-actin were obtained, which were represented as relative to 100 % DMSO.

Hitzman R.T., Dunlap T.L., Howell C.E., Chen S.N., Vollmer G., Pauli G.F., Bolton J.L, & Dietz B.M. (2020). 6-Prenylnaringenin from Hops Disrupts ERα-mediated Downregulation of CYP1A1 to Facilitate Estrogen Detoxification. Chemical research in toxicology, 33(11), 2793-2803.

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Western Blot Analysis of Protein Expression

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To analyze protein expression, cells were lysed in radioimmunoprecipitation assay (RPPA) buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA) supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland). The same amount of proteins was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes using the iBlot gel transfer device (Thermo Fisher Scientific). The membranes were blotted with appropriate primary antibodies and horseradish peroxidase (HRP)–conjugated secondary antibody to detect the proteins of interest. The monoclonal anti-vimentin antibody 84–1 was produced in a mouse. The anti-RPS6-p235p236, anti-RPS6-p240p245, anti-p70S6K-p371, anti-p70S6K-p389, anti-p70S6K, anti-pERK, anti-ERK, anti-p-mTOR, anti-mTOR, and anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primary antibodies were from Cell Signaling Technology (Danvers, MA). The anti-mouse and anti-rabbit horseradish peroxidase (HRP)–conjugated secondary antibodies were from Santa Cruz Biotechnology (Dallas, TX).

Noh H., Yan J., Hong S., Kong L.Y., Gabrusiewicz K., Xia X., Heimberger A.B, & Li S. (2016). Discovery of cell surface vimentin targeting mAb for direct disruption of GBM tumor initiating cells. Oncotarget, 7(44), 72021-72032.

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Western Blot Analysis of Liver Proteins

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Distinct Liver samples (n = 3) were homogenized using bead crushers µT−12 (TAITEC) in RIPA buffer supplemented with a 1× protease inhibitor cocktail (both from Nacalai Tesque). Total proteins were quantitated using a bicinchoninic acid protein assay (Takara Bio Inc.), and 50 µg total protein was added to 1 M DTT and heated to 95 °C for 3 min for thermal denaturation. Proteins were separated on a 4–20% gradient SDS-PAGE gel and transferred to a nitrocellulose membrane using an iBlot Gel Transfer Device (Thermo Fisher Scientific). Membranes were blocked with Blocking One (Nacalai Tesque) at room temperature for 30 min and incubated with primary antibodies in blocking buffer at room temperature for 1 h or at 4 °C overnight. After washing thrice for 10 min with PBS-T, the membranes were incubated with secondary antibodies in blocking buffer at room temperature for 1 h. Then, membranes were washed thrice with PBS-T, and proteins were detected using enhanced chemiluminescence (Thermo Fisher Scientific). Images were collected using the Bio-Rad Molecular Imager ChemiDoc Touch (Supplementary Fig. 14).

Terada C., Oh K., Tsubaki R., Chan B., Aibara N., Ohyama K., Shibata M.A., Wada T., Harada-Shiba M., Yamayoshi A, & Yamamoto T. (2023). Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry. Nature Communications, 14, 7972.

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Western Blot and Dot Blot Analysis

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Cell extracts were isolated in RIPA buffer supplemented with 1 × Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and were separated on a 4–12% gradient SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane using iBlot Gel Transfer Device (Thermo Fisher Scientific). The membranes were blocked at room temperature for 30 min with blocking buffer containing 5% (w/v) nonfat dry milk in 1 × PBS and incubated with primary antibodies in blocking buffer at 4°C overnight. After washing three times with washing buffer (0.1% Tween-20 in 1 × PBS) for 5 min each wash, membranes were incubated with secondary antibodies in blocking buffer at room temperature for 1 h. After washing three times with washing buffer for 5 min each wash, proteins were detected based on Enhanced chemiluminescence (Abcam) using ChemiDoc XRS+ (Bio-Rad). The signal intensities of the protein bands were measured and quantitated using Image Lab software (Bio-Rad). Antibodies for western blotting used in this study are listed in Supplemental Table S1. For dot blots, 5 μg of purified genomic DNA was treated with E. coli RNase H (NEB) for 20 min at 37°C. Treated or untreated DNA samples were spotted onto a nitrocellulose membrane. DNA:RNA hybrids were detected using the S9.6 antibody. Total DNA was detected by SYBR™ Gold (Thermo Fisher Scientific).

Shen W., Sun H., De Hoyos C.L., Bailey J.K., Liang X.H, & Crooke S.T. (2017). Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops. Nucleic Acids Research, 45(18), 10672-10692.

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Protein Quantification by Western Blot

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Samples (15 μg protein/lane) were electrophoresed on SDS‐PAGE, next, transferred onto polyvinylidene difluoride membranes by iBlot Gel Transfer Device (Thermo Fisher). The membranes were blocked by Blocking One (Nacalai Tesque, Kyoto, Japan), thereafter, incubated with primary antibodies at 4°C overnight. Afterward, these protein bands were incubated by horseradish peroxidase (HRP)‐conjugated secondary antibodies (Cell Signaling Technology). Bands were treated with ECL Prime Western Blotting Detection Reagents (GE Healthcare Life Sciences, Little Chalfont, UK). Finally, ImageQuant TL GE Healthcare Life Sciences) was used to digitize the band strength.

Luo J., Zhu H., Jiang H., Cui Y., Wang M., Ni X, & Ma C. (2018). The effects of aberrant expression of LncRNA DGCR5/miR‐873‐5p/TUSC3 in lung cancer cell progression. Cancer Medicine, 7(7), 3331-3341.

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Western Blot Analysis of Protein Expression

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Western blots were carried out as detailed previously (7 (link)). Cells in 6 cm plates were transfected with NLuc-fusion plasmids (500 ng) using Effectene transfection reagent (Qiagen). Following a 24-h incubation, cells were removed from the plate by trypsinization, pelleted, washed 1× with PBS, then resuspended in IP Lysis buffer (Pierce) supplemented with 1× Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), quantitated using a BCA protein assay (Pierce), and separated on a 4–12% gradient SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane using iBlot Gel Transfer Device (Thermo Fisher Scientific). The membranes were blocked at room temperature for 30 min with blocking buffer containing 5% (w/v) nonfat dry milk in 1× PBS and incubated with primary antibodies in blocking buffer at room temperature for 2 h or 4°C overnight. After washing three times with washing buffer (0.1% Tween-20 in 1× PBS) for 5 min each wash, membranes were incubated with secondary antibodies in blocking buffer at room temperature for 1 h. After washing three times with washing buffer for 5 min each wash, proteins were detected based on enhanced chemiluminescence (Abcam).

Vickers T.A., Rahdar M., Prakash T.P, & Crooke S.T. (2019). Kinetic and subcellular analysis of PS-ASO/protein interactions with P54nrb and RNase H1. Nucleic Acids Research, 47(20), 10865-10880.

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Western Blot Analysis of Cell Lysates

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Compounds dissolved in DMSO were assayed by Western blot analysis. The cells were washed with phosphate buffered saline (PBS) and then treated with the lysis buffer. The cellular lysates were centrifuged at 13,800× g for 5 min. The total cellular extracts were separated on SDS-polyacrylamide gels (4–12% SDS-polyacrylamide; Thermo Fisher Scientific K.K, Yokohama, Japan) and transferred to a nitrocellulose membrane (iBlot Gel Transfer Mini; Thermo Fisher Scientific K.K, Yokohama, Japan ) using an iBlot Gel Transfer Device (Thermo Fisher Scientific K.K, Yokohama, Japan). Protein detection was carried out using an immunodetection system (Invitrogen, Thermo Fisher Scientific K.K, Yokohama, Japan) with the antibodies. The protein concentration of the cells was determined using a BCA protein assay kit (Thermo Fisher Scientific K.K, Yokohama, Japan).

Taira J., Miyazato H, & Ueda K. (2018). Marine Peroxy Sesquiterpenoids Induce Apoptosis by Modulation of Nrf2-ARE Signaling in HCT116 Colon Cancer Cells. Marine Drugs, 16(10), 347.

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