Western blot (WB) analysis was performed according to standard procedures. The antibodies used in these experiments are listed in Table S2
Eblot l1
Manufactured by GenScript
Sourced in United States
The EBlot L1 is a compact and efficient electroblotting system designed for the transfer of proteins from polyacrylamide gels to membranes. It features a simple and user-friendly operation with adjustable voltage and run time settings to accommodate a variety of transfer requirements.
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Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Surepage, by GenScript (1 mentions)
Ha1006, by Huabio (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Ab154141, by Abcam (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Any kd precast gel, by Bio-Rad (1 mentions)
Image studio software, by LI COR (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Ab15348, by Abcam (1 mentions)
Ab92323, by Abcam (1 mentions)
Nitrocellulose membrane, by Advansta (1 mentions)
Ab49900, by Abcam (1 mentions)
Complete mini edta free protease inhibitor, by Merck Group (1 mentions)
Hpa000288, by Merck Group (1 mentions)
9 protocols using eblot l1
1
Comprehensive Western Blot Analysis Protocol
2
Co-Immunoprecipitation Assay of Plant Proteins
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Co‐immunoprecipitation (Co‐IP) assays were performed as described previously (Zhang et al., 2019 (link)). About 0.5 g of agroinfiltrated leaf tissue was frozen in liquid nitrogen, ground to a fine powder, and then thawed in a plant protein extraction buffer (100 mM Tris–HCl, pH 8.8, 60% SDS, and 2% B‐mercaptoethanol) with protease inhibitor cocktail tablets (one tablet per 50 mL buffer). The mixture was centrifuged at 18 000 g for 10 min at 4 °C. Each supernatant (500 μL) was mixed with 45 μL of anti‐GFP‐conjugated agarose beads (Sigma) and incubated at 4 °C for 1.5 h with gentle shaking. Agarose beads were pelleted and washed three times with the Co‐IP buffer. The resulting pellets were boiled in SDS loading buffer. For the immunoblot, proteins were separated on 10% SDS‐PAGE gels (SurePAGE™, Genscript, M00652) through electrophoresis, and then transferred to NC membranes using eBlot™ L1 (Genscript, L00686C). The blots were probed with an anti‐HA (1 : 5000) and an anti‐GFP (1 : 5000), followed by an HRP‐conjugated secondary antibody (HUABIO, HA1006).
3
Western Blot Analysis of MK-STYX Transfection
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HEK/293 cells were transfected with pMT2, pMT2-FLAG-MK-STYX-FLAG, GFP, mCherry, GFP-MK-STYX, or mCherry-MK-STYX expression plasmids, then lysed and analyzed by Western blotting. Cells were harvested in lysis buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, 1% Nonidet P-40 alternative (Calbiochem, Burlington, MA, USA), and protease inhibitor cocktail tablets (Roche, Branchburg, NJ, USA). Lysates were sonicated, centrifuged at 14,000× g for 10 min, and the supernatant protein concentration was determined by NanoDrop quantification. Lysates were resolved by 4%–20% Bis-Tris gels and transferred to PVDF by the eBlot L1 (Genscript, Piscataway, NJ, USA). Chemiluminescence was detected using a BioRad ChemiDoc MP imaging system for immunoblot analysis with anti-phospho-HDAC6 (pSer22); anti-HDAC6; anti-phosphotyrosine, clone 4G10; anti-acetylated-lysine; anti-STYXL1; anti-GAPDH; anti-β-tubulin polyclonal; monoclonal acetylated microtubule; anti-β-tubulin, detyrosinated; anti-GFP; and monoclonal anti-mCherry antibodies, followed by chemiluminescent detection. When warranted, blots were stripped (200 mM glycine, 3.5 mM SDS, 1% Tween 20) and re-probed.
4
Western Blot Analysis of FUS Protein
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Lysates were mixed with 4× NuPAGE LDS Sample Buffer (Thermo NP0008) and run through AnyKD precast gels (BioRad 4569034) at 80 V for 2 h. Gels were transferred through either a Trans-Blot Turbo Transfer System (Bio-Rad 1704150) or eBlot L1 (GenScript L00686) onto nitrocellulose membranes (BioRad 1620112). Membranes were blocked with 6% milk (BioRad 1706404) in Tris-buffered saline (TBS). Primary and secondary antibodies were diluted in TBS with 0.1% Tween-20 (Sigma P7949). The following primary antibodies were used to probe the blots: FUS antibodies (Santa Cruz 373698, Abcam ab154141, Bethyl A300-293A, custom rabbit phospho-FUS antibodies), gamma tubulin (Sigma T6557), and GFP (Roche 11814460001). Primary antibodies were detected with secondaries conjugated to IRDye fluorescent probes (LI-COR 926-68021, 926-32210). Blots were imaged with the Odyssey CLx imaging system (LI-COR). Band densitometry quantification was done using Image Studio software (Li-COR). Phosphobands were normalized to endogenous FUS band intensity.
5
Protein Expression and Quantification
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Cells were lysed using RIPA buffer (Thermo Fisher Scientific) containing cOmplete, Mini, EDTA-free protease inhibitor (Sigma-Aldrich). Protein concentration was determined by BCA Protein Assay kit (Thermo Fisher Scientific). 10 µg/sample with 4x Laemmli Buffer (Bio-Rad, Hercules, CA, USA) containing β-mercaptoethanol was resolved in 20% Mini-PROTEAN TGX Gel (Witec AG, Sursee, Switzerland). PNGase treatments have been performed according to the manufacturer’s protocol (New England BioLabs, Frankfurt, Germany). Gels were transblotted on nitrocellulose membrane (Advansta) using eBlot L1 (GenScript, Piscataway, NJ, USA). Membranes were blocked and stained with primary antibody overnight in 5% nonfat dry milk in 0.1% phosphate-buffered saline Tween, visualized with horseradish peroxidase (HRP)-conjugated secondary antibody using WesternBright Quantum (Advansta Corporation, Menlo Park, CA, USA) and imaged by Fusion FX 7 Imaging System (Vilber Lourmat, Eberhardzell, Germany). Antibodies used are rabbit anti-ACE2 (1:500, ab15348, Abcam, Cambridge, UK), rabbit anti-ACE2 (1:1000, HPA000288, Sigma-Aldrich), rabbit anti-TMPRSS2 (1:1000, ab92323, Abcam), rabbit anti-NRP1 (1:50, HPA030278, Sigma-Aldrich), HRP-conjugated anti-rabbit (1:10000, 11-035-003, Jackson), and HRP-conjugated anti β-actin (1:25000, ab49900, Abcam).
6
Protein Immunoblotting of Eimeria Antigens
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Following resolution by 10% SDS-PAGE, each purified protein sample was transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) using eBlot™ L1 (GenScript, Nanjing, China). The membranes were separately incubated at 37 °C for 2 h with mouse anti-His tag monoclonal antibody (CWBIO, Beijing, China), chicken anti-E. tenella serum, and normal chicken serum. After washing with TBST buffer, the membranes were separately incubated at 37 °C for 1 h with the corresponding secondary antibodies: peroxidase-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-chicken IgY (Abcam, Cambridge, UK). The binding of secondary antibodies was revealed by the enhanced HRP-DAB chromogenic substrate kit (TIANGEN, Beijing, China).
Protein extracts of merozoites were obtained by using RIPA lysis buffer (Beyotime, Nantong, China). Western blot analysis was carried out using mouse anti-rEtROP17 serum as the primary antibody. Goat anti-mouse IgG conjugated with horseradish peroxidase (Abcam, Cambridge, UK) was used as a secondary antibody. The bound antibody was detected using protein enhanced chemiluminescent (ECL) reagent (Thermo Scientific, Waltham, MA, USA). The slide stained with the serum before immunization as the primary antibody was used as the control.
Protein extracts of merozoites were obtained by using RIPA lysis buffer (Beyotime, Nantong, China). Western blot analysis was carried out using mouse anti-rEtROP17 serum as the primary antibody. Goat anti-mouse IgG conjugated with horseradish peroxidase (Abcam, Cambridge, UK) was used as a secondary antibody. The bound antibody was detected using protein enhanced chemiluminescent (ECL) reagent (Thermo Scientific, Waltham, MA, USA). The slide stained with the serum before immunization as the primary antibody was used as the control.
7
Hep3B and CAR T Cell Co-culture
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Hep3B cells (0.5 × 106) and CAR T cells were cocultured at the indicated time intervals and cell lysates were harvested. The gel and membranes were run using standard protocols for wet transfer at room temperature (Genscript eBlot L1). The membranes were stained and probed overnight at 4°C using the antibodies listed in the Supplementary Data (Table S1, http://links.lww.com/HC9/A76 ).
8
Protein Extraction and Western Blot Analysis of L. striatellus
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The organs of 300 L. striatellus were dissected on iced 1× PBS buffer with tweezers, under an optical microscope, and immediately lysed with RIPA (radio immunoprecipitation assay) lysis buffer (P0013B, Beyotime Biotec.). The induced cells were centrifuged at 12,000 rpm for 2 min. All samples were suspended with 5× loading buffer (2.5 mM Tris-HCl, 0.1% w/v SDS, 0.005% w/v BPB, 0.4% w/v glycerin, 500 mM DTT, pH 6.8), boiled for 10 min and then detected with SDS–polyacrylamide gel electrophoresis (PAGE). The gels were then stained with Coomassie Brilliant Blue R250. After SDS–PAGE, the proteins were transferred to PVDF membranes by eBlotTM L1 (Genscript) and incubated with Ls-TRPML polypeptide primary antibody (produced by Genscript, 1:2000) or MBP Rabbit Monoclonal Antibody antibody (AF1225, Beyotime Biotec., 1:1000), followed with HRP conjugated secondary antibody (Beyotime Biotec., 1:1000). The blotted membranes were visualized by using a chemiluminescence gel imaging system.
9
Quantitative Yeast Cre Protein Analysis
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Yeast cell pellets (10 OD600 units) were resuspended and incubated with 200 µl NaOH (100 mM) at room temperature for 5 min. The NaOH-treated yeast cells were harvested via centrifugation (10,000 g for 2 min) and were subjected to cell lysis treatment via boiling for 10 min in 100 µl of 2x SDS-PAGE loading buffer (Beyotime, Cat number: P0015B). Equivalent levels of protein loading for whole cells were determined on the basis of the OD600 of the cell culture (0.1 units per lane) and confirmed by Coomassie's brilliant blue staining of parallel gels. Proteins were separated by reducing 12 % SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore) via eBlotTM L1 (Genscript) transfer system. Cre enzymes were detected by the Cre-specific monoclonal antibody (Invitrogen, Cat number: MA5-27870) and alkaline phosphatase-linked anti-mouse IgG peroxidase antibody (Sigma-Aldrich, Cat number: A2304). Immunoreactive antigens were detected by chemiluminescence using horseradish peroxidase (HRP) substrate (Millipore, Cat number: WBKLS0100).
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