Protein samples were mixed with SDS gel loading buffer (Quality Biologicals, Gaithersburg, MD) and heated for 10 minutes at 70°C. Samples were loaded onto a 4–12% polyacrylamide gel (NuPAGE BT, Life Technologies, Carlsbad, CA) and fractionated according to manufacturer’s instructions. Protein bands were visualized by staining with Coomassie brilliant blue R-250 (Sigma). The protein concentrations were measured using the Bradford assay (Bio-Rad). For the Western blot, protein samples were transferred from the polyacrylamide gel to a PVDF membrane using the Pierce G2 fast blotter (Thermo Scientific, Rockford, IL) for 10 minutes. The membrane was blocked with 5% non-fat dry milk in PBS/0.01% Tween 20 for 1 h at 37 °C, washed three times with PBS/0.01% Tween 20, and then incubated with DENV-2 hyperimmune mouse ascitic fluid (HMAF) at 1:100 in blocking buffer for 1 h at 37 °C. After washing, the secondary antibody was peroxidase-conjugated goat anti-human IgG (Kirkegaard & Perry, Gaithersburg, MD) diluted in blocking solution and incubated for 1 h at 37 °C. The membrane was washed again and the bands were detected by incubation with 3, 3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System (Kirkegaard & Perry).
Pierce g2 fast blotter
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce G2 Fast Blotter is a compact and efficient device designed for transferring proteins from polyacrylamide gels to membrane supports during Western blotting procedures. The device utilizes a semi-dry transfer method to facilitate rapid and reliable protein transfer, enabling researchers to obtain high-quality blotting results in a timely manner.
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Lab products found in correlation
Odyssey blocking buffer, by LI COR (5 mentions)
Image studio software, by LI COR (4 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (3 mentions)
Nupage gel, by Thermo Fisher Scientific (2 mentions)
Dylight secondary antibodies, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
A0408, by Merck Group (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Hpa alexa 647, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
4 15 gradient gels, by Thermo Fisher Scientific (2 mentions)
Complete edta free protease inhibitor, by Merck Group (2 mentions)
Mini protean precast gel, by Bio-Rad (2 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Hyperfilm ecl, by GE Healthcare (1 mentions)
Trans blot cell, by Bio-Rad (1 mentions)
Protran nitrocellulose membrane, by GE Healthcare (1 mentions)
37 protocols using pierce g2 fast blotter
1
SDS-PAGE and Western Blot Analysis of DENV-2 Proteins
2
Western Blotting: Quantitative Protein Analysis
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Samples were separated on poly-acrylamide gels and transferred to Protran nitrocellulose membranes (0.45 μm, GE Healthcare) using a Pierce G2 Fast Blotter (Thermo Scientific) or Bio-Rad TransBlot cell. Membranes were blocked for 1 h at room temperature in TBS containing 5% milk powder/0.1% Tween-20 (TBST-M) and probed with primary antibodies in TBST-M for either 1 h at room temperature or 16 h at 4 °C. After washing with TBS/0.1% Tween-20 (TBST), membranes were incubated with horseradish peroxidase (HRP)-conjugated or alkaline phosphatase (AP)-coupled, species-specific secondary antibodies. Following incubation with SuperSignal West Pico Chemiluminescence Substrate (Thermo Scientific) or AttoPhos® AP Fluorescent Substrate (Promega) signals were detected on Hyperfilm ECL (GE Healthcare) or using a G: Box imager (Syngene). Films were scanned on a CanoScan LiDE 60 photo scanner and signals were quantified using the Fiji distribution (http://fiji.sc/ ) (72 (link)) of the ImageJ image processing software [National Institutes of Health (NIH), http://rsb.info.nih.gov/ij /] (73 (link)).
3
Fluorescent Lectin Blotting Optimization
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To perform blots with fluorescent lectins, 20 µg of cell lysates or secretome samples were loaded onto Bio-Rad (4–15%) gradient gels, and proteins were transferred to nitrocellulose membrane using the Thermo Scientific Pierce G2 Fast Blotter. The membranes were blocked with Odyssey blocking buffer (LI-COR) for 20 min. Helix Pomatia Agglutinin (HPA)-Alexa 647 (Thermo Fisher) or Galanthus Nivalis Lectin (GNL) conjugated to Alexa 647 were diluted 1 : 1,000 in Odyssey blocking buffer from their stock concentration of 1 µg/µl and 5 µg/µl, respectively. Membranes were incubated with diluted HPA-Alexa 647 or GNL-Alexa 647 for 90 min and imaged using the Odyssey Imaging System.
4
Cell Cycle Synchronization and Protein Analysis
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For cell cycle synchronization, the cultures were grown to OD600 = 0.3 at 30 °C, followed by addition of α-factor at final concentration of 1 mg/L. The cultures were grown for 2.5 h, when the cells were pelleted, washed thoroughly and resuspended in medium without α-factor to release the cells to the cell cycle. At the indicated time point, cells were pelleted and snap-frozen. The cells were resuspended in urea lysis buffer and were lysed by bead beating. After clearing the lysate by centrifugation, the total protein concentration was measured with Bio-Rad Protein Assay Dye Reagent. Cln2-7myc and Whi7-13myc samples were loaded on 8% acrylamide SDS-PAGE gels. Cdc24-13myc phosphorylation was analyzed using 6% acrylamide Mn2+-Phos-tag SDS-PAGE gels supplemented with 25 µM Phos-tag [53 (link)]. The proteins were transferred to nitrocellulose membranes using Pierce G2 Fast Blotter (ThermoFisher Scientific, Waltham, MA, USA). For loading control, the membranes were stained with Ponceau S dye. Rabbit polyclonal c-myc antibody (sc-789, Santa Cruz Biotechology, Dallas, TX, USA) and HRP-conjugated anti-rabbit IgG antibody (1:7500) from Labas, Estonia were used for detection of myc-tagged proteins.
5
Western Blot Analysis of DNA Damage Signaling
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Cells were seeded in 6-well plates and allowed to attach for 24 h. Cells were then incubated for 24 hours with indicated concentrations of AZ31 and/or SN38. Cells were then washed with PBS and lysed with RIPA buffer (Cell Signaling, Danvers, MA). After sonication and centrifugation, a total of 30 μg of protein lysate was loaded onto a NuPage gel (Life Technologies, Carlsbad, CA), electrophoresed, and transferred to a nitrocellulose membrane using the Pierce G2 FastBlotter (Thermo Fisher, Rockford, IL). The membrane was blocked and probed overnight with primary antibodies (Cell Signaling Technologies (Danvers, MA) at a concentration 1:1000: p-ATM (catalog# 13050), ATM (catalog# 2873), p-p53 (catalog# 9284), p53 (catalog# 2527), p-CHK2 (catalog# 2665), p-RAD50 (catalog# 14223), p-H2AX (catalog# 9718) and actin (catalog# 4970). The next day the membranes were washed for 10 minutes 3X with TBS/Tween 20, and then probed with DyLight secondary antibodies 1:15,000 (Cell signaling, Danvers, MA), and imaged using the Licor Odyssey (Licor, Lincoln, NE).
6
Asynchronous and Synchronized Yeast Cultures
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For analysis of asynchronous cultures, Ypr174c-6HA expressing cells were grown in 5 mL YPD at 30°C to OD 0.8, collected by centrifugation and flash-frozen. For synchronized metaphase release, PGALS-CDC20 cells were grown in YP Raf+Gal to OD 0.3, then cells were collected and resuspended in YP Raf. Cells were grown for 3 hours, resulting in over 90% of cells being in metaphase, then released to anaphase by addition of 2% galactose. For lysis, cells were resuspended in urea lysis buffer and disrupted by bead beating. The lysates were separated using SDS-PAGE and proteins were transferred to nitrocellulose membranes using Pierce G2 Fast Blotter (Thermo Scientific). anti-HA.11 epitope tag antibody (1:500) (clone 16B12, BioLegend Cat. No. 901501) and HRP-conjugated anti-mouse IgG antibody (1:7,500) from Labas, Estonia were used to detect Ypr174c-6HA. For detection of Clb2, rabbit polyclonal Clb2 antibody (1:500) (y-180, Santa Cruz Biotechnology) and HRP-conjugated anti-rabbit antibody (1:7500) from Labas, Estonia were used. All western blottings were performed in at least two biological replicates.
7
Western Blot Analysis of Rac1
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Total extracts of respective cell lines were obtained by washing cells three times with ice cold PBS (phosphate buffered saline), addition of 4x Laemmli buffer and scraping cells. Extracts were boiled 4 minutes at 95 °C and separated by SDS-PAGE. Samples were transferred to PVDF membrane using Pierce G2 Fast Blotter (Thermo Scientific). After blocking in 10% dry milk in TBS-T, the membrane was incubated with Rac1 antibody (23A8, 1:1000, Merck Millipore) and peroxidase conjugated goat anti mouse antibody. Membrane was developed with LumiLight (Roche) using an Imager (ECL ChemoCam HR3.2, Intas). PVDF membrane was stained with Coomassie solution (0.1% Coomassie brilliant blue R-250 in 50% methanol, 7% acetic acid) afterwards and dried. Data were processed with Fiji and Photoshop CS6.
8
Immunoprecipitation and Western Blotting of FcεRI Signaling
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BMMCs or MC/9 were stimulated as described above. Cells were lysed in 1% Nonidet-P-40 lysis buffer (20 mM Tris-HCl, pH 7.5, and 150 mM NaCl) supplemented with AEBSF, aprotinin, leupeptin, pepstatin, sodium fluoride, sodium orthovanadate, and β-glycerophosphate on ice for 20 min. Lysates were centrifuged at 4°C for 15 min. For Western blotting analysis, SDS-containing sample buffer was added to lysates before loading onto 10 or 12% SDS-PAGE gels when analyzing for FcεRI β and γ chains. For IPs, lysates were incubated overnight at 4°C with appropriate antibodies. Immune complexes were precipitated at 4°C for 2 h with protein A or G agarose beads (Thermo Fisher Scientific) for rabbit or mouse Abs, respectively, then washed with NP-40 lysis buffer three times before analysis by SDS-PAGE gels. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes using the PierceG2 fast blotter (Thermo Fisher Scientific), probed with appropriate antibodies, and imaged on a ProteinSimple Fluochem M cooled charge-coupled device imager (ProteinSimple).
9
Alisertib Dose-Dependent Protein Analysis
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Cells were seeded in 6-well plates and allowed to attach for 24h. Cells were then incubated for 8, 12, 24h or 48h with varying concentrations of alisertib. Cells were then washed with PBS and lysed with RIPA buffer (Cell Signaling, Danvers, MA). After sonication and centrifugation, a total of 30 μg of protein lysate was loaded onto a NuPage gel (Life Technologies, Carlsbad, CA), electrophoresed, and transferred to a nitrocellulose membrane using the Pierce G2 FastBlotter (Thermo Fisher, Rockford, IL). The membrane was blocked and probed overnight with primary antibodies, washed for 10 minutes 3X with TBS/Tween 20, and probed with DyLight secondary antibodies (Cell signaling, Danvers, MA), and imaged using the Licor Odyssey (Licor, Lincoln, NE). All primary antibodies were purchased from Cell Signaling Technology (Danvers, MA) and diluted as per the manufactures' instructions.
10
Quantitative Western Blot Analysis of Secreted MYOC
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For all Western blot-related experiments on secreted MYOC, 2% FBS containing media was used to minimize gel warping and epitope masking due to excessive FBS/BSA. Conditioned media (20 μL) was denatured in either reducing or nonreducing Laemmli buffer at 95°C for 5 minutes and loaded onto a Tris-Gly SDS-PAGE gel. Proteins were separated and transferred onto a nitrocellulose membrane using a Pierce G2 Fast Blotter (Thermo Scientific). Membranes then were blocked in Odyssey blocking buffer (LI-COR, Lincoln, NE, USA), and probed with either a goat anti-MYOC antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), a rabbit anti-GLuc antibody (1:2000; NEB), a mouse anti-FLAG M2 antibody (1:2000, Sigma-Aldrich Corp.), a rabbit anti-GRP94 (1:1500, GeneTex, Irvine, CA, USA), or a mouse anti-β-actin (1:10,000, Sigma-Aldrich Corp.) primary antibody followed by an appropriate near-infrared secondary antibody (1:10,000; LI-COR). A LI-COR Odyssey Fc infrared imager (LI-COR) was used to image the blots, and the Image Quant software was used to quantify protein bands.
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