Tgx stain free sds page gel

Manufactured by Bio-Rad

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The TGX Stain-Free SDS-PAGE gel is a precast polyacrylamide gel used for separating proteins based on their molecular weight. It allows for the visualization of proteins without the need for traditional staining methods.

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7 protocols using tgx stain free sds page gel

Western Blot Analysis of Myc-Tagged Proteins

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Yeast cells were grown as described as for polysome assays. Lysates were prepared as described by Zhang et al^{93 (link)} An equal volume (10 μL) of each sample was loaded in a 10% TGX stain-free SDS-PAGE gel (Bio-Rad 161–0183). Proteins were transferred using the Trans-Blot Turbo Transfer System (Bio-Rad 1704150) and ready-to-assemble transfer kits (Bio-Rad 1704273). The manufacturer recommended time (3 min) was used for transfer.
The membrane was blocked overnight at 4°C in tris-buffered saline with 20% Tween-20 (TBST) and 5% skim milk (Bioshop SKI400.500). A primary rabbit anti-myc-tag polyclonal antibody (PA1–981) and a primary mouse anti-GAPDH antibody dilution (1:5000) were prepared in TBST with 5% skim milk. The membrane was incubated in both primary rabbit and mouse antibody simultaneously for an hour at room temperature. The membrane was washed 3 times in TBST for 5 min each. The membrane was then incubated at room temperature for an hour with a secondary rabbit and mouse HRP antibody simultaneously (1:5000 dilution) in TBST. A Bio-Rad Gel Doc System was used to image the membrane after 3 washes in TBST for 5 minutes each.

Lu H., Mazumder M., Jaikaran A.S., Kumar A., Leis E., Xu X., Altmann M., Cochrane A, & Woolley G.A. (2019). A yeast system for discovering optogenetic inhibitors of eukaryotic translation initiation. ACS synthetic biology, 8(4), 744-757.

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Western Blot Analysis of IL-17RB

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Cell debris from protein lysates was removed by centrifugation at 9000×g for 10 min at 4 °C and protein concentration was determined by BCA assay (ThermoFisher, USA). Five micrograms of protein was resolved by 4–15% TGX Stain-free SDS PAGE gel (Biorad, USA) and semi-dry transferred onto nitrocellulose (Biorad, USA). Membranes were blocked in 3% BSA 2% skim milk in TBS-T for 1 h at room temperature, then incubated overnight at 4 °C with anti-IL-17RB or β-actin (ab8227, Abcam). After extensive washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 h. Membranes were developed with Supersignal west femto (ThermoFisher, USA) and imaged on a Chemi-Doc (Biorad, USA).

Williams T.C., Loo S.L., Nichol K.S., Reid A.T., Veerati P.C., Esneau C., Wark P.A., Grainge C.L., Knight D.A., Vincent T., Jackson C.L., Alton K., Shimkets R.A., Girkin J.L, & Bartlett N.W. (2022). IL-25 blockade augments antiviral immunity during respiratory virus infection. Communications Biology, 5, 415.

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Quantitative Western Blotting for SARS-CoV-2 Proteins

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Western blotting was performed as described (MacCallum et al., 2006 (link); Chan, 2016 (link); Mufrrih et al., 2021 (link)). Protein lysates were harvested into 2xSDS-PAGE loading buffer (0.125 M Tris pH6.8, 4% SDS, 10% β-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue). Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer. Anti-ACE2 antibody (Proteintech) was used at 1:2000 and anti-mouse HRP (Cell Signaling Technology) at 1:1,000. Anti-SARS-CoV-2 spike antibody (BEI Resources NR-52947) was used at 1:1,000 and anti-rabbit HRP (Cell Signaling Technology) at 1:1,000. Protein bands were detected using Clarity™ ECL substrate (Bio-Rad). Images were captured and quantified using ChemiDoc™ XRS + system (Bio-Rad) and ImageLab 6.0.1 software (Bio-Rad).

, & Chan S.W. (2022). Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation. Frontiers in Pharmacology, 13, 1007527.

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Membrane Protein Extraction and Analysis

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Tissue samples were homogenized in Hank’s balanced salt solution containing complete protease inhibitor cocktail (1 tablet/10 mL, Roche, Indianapolis, IN) and phosphatase inhibitor cocktail (1:100; Sigma). After centrifugation (10 000g; 15 min at 4°C), the supernatant was collected, and the membrane protein fraction prepared by suspending pellets in lysis buffer (0.3M NaCl, 50 mM Tris-HCl pH7.6, and 0.5% Triton X-100) with protease/phosphatase inhibitors as above. Supernatants were pooled for whole cell lysates and protein concentrations determined using a BCA protein assay (Pierce, Rockford, IL). After denaturation (100°C for 5 min) in Laemmli sample buffer, each lysate was separated on a 4–15% TGX Stain-Free SDS-PAGE gel (Bio-Rad, Hercules, CA). Proteins were transferred to PVDF membranes and incubated overnight at 4°C with primary antibodies against RXFP1, RXFP2 and smooth muscle actin (details of antibodies in Supplemental Figure S11) diluted in Tris-buffered saline with 0.1% Tween-20 (TBS-T) containing 5% (w/v) milk. Membranes were incubated with appropriate horseradish peroxidase conjugated secondary antibodies in 5% (w/v) Milk TBS-T, washed, and incubated in WesternBright Quantum (Advansta, Menlo Park, CA) for chemiluminescent imaging (ChemiDoc MP, Bio-Rad). Optical density of each protein species was normalized to total protein levels using Image Lab software (Bio-Rad).

Ikeda Y., Zabbarova I.V., Birder L.A., Wipf P., Getchell S.E., Tyagi P., Fry C.H., Drake M.J, & Kanai A.J. (2018). Relaxin-2 therapy reverses radiation-induced fibrosis and restores bladder function in mice. Neurourology and urodynamics, 37(8), 2441-2451.

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SDS-PAGE and Western Blot Analysis of Yeast VLPs

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Yeast cell lysate supernatants or purified VLP samples were diluted in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled at 100 °C for 10 min, electrophoresed through 10% TGX Stain-Free SDS-PAGE gel (Bio-Rad, Hercules, CA, USA) and proteins were detected by Stain-Free technology (Bio-Rad, Hercules, CA, USA). Then gel was transferred to a PVDF membrane using a Bio-Rad semi-dry apparatus. The membrane was blocked with 5% skim milk in TBS-T and probed with the anti-L1 mAb [CamVir 1] (Abcam, Cambridge, UK) at dilution of 1:2000, and a secondary antibody directed against mouse IgG conjugated to Peroxidase. Bound antibodies were be detected using an enhanced chemiluminescence detection kit (GE Health care, Chicago, IL, USA).

Eto Y., Saubi N., Ferrer P, & Joseph-Munné J. (2021). Expression of Chimeric HPV-HIV Protein L1P18 in Pichia pastoris; Purification and Characterization of the Virus-like Particles. Pharmaceutics, 13(11), 1967.

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Retinal Protein Immunoblotting Assay

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Following euthanasia eyes were removed, and the retina was dissected from the eye and snap frozen on dry ice. Single retinas (n = 3 per genotype) were homogenized independently in RIPA buffer (150 mM NaCl, 50 nM Tris, pH 8.0, 1% IGEPAL, 0.5% Deoxycholate, 0.1% SDS) containing 1X protease inhibitors (Roche). Lysate concentration was determined using a Pierce BCA protein assay kit (Thermo) and samples were equally loaded and separated on a 4–15% TGX Stain-Free SDS-PAGE gel (Bio-Rad). Following electrophoresis, separated proteins were wet transferred to PVDF for immunoblotting. Blots were incubated in StartingBlock PBS blocking buffer (Thermo) for >1 hr prior to incubation with antibodies diluted in blocking buffer. After overnight incubation in primary antibody, blots were washed several times in PBS before incubation with secondary antibody for approximately 1 h. After several PBS washes, blots were incubated with substrate (SuperSignal West Pico, Thermo) and digitally imaged on a Bio-Rad ChemiDoc imaging system.

Gumerson J.D., Alsufyani A., Yu W., Lei J., Sun X., Dong L., Wu Z, & Li T. (2021). Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing. Gene Therapy, 29(1-2), 81-93.

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SARS-CoV-2 Spike Protein Expression Analysis

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HEK293T cells were transiently transfected in 6-well plates with 1.6 µg of pcDNA3.1, pcDNA3.1 S.dTM.PP or pcDNA3.1 S.dTM.PP-CD40L using Lipofectamine™ 3000 Transfection Reagent (ThermoFisher, Ottawa, ON) according to the manufacturer’s instructions and incubated for 48 hours at 37°C, 5% CO₂. The cells were washed with phosphate-buffered saline (PBS) and then lysed with radioimmunoprecipitation assay buffer (ThermoFisher, Ottawa, ON). Lysates were electrophoresed on a 4-15% TGX stain-free SDS-PAGE gel (Bio-Rad, Saint-Laurent, QC) and subsequently transferred to a polyvinylidene difluoride membrane. Membranes were blocked for 1h at room temperature with tris-buffered saline (TBS) containing 0.5% Tween 20 (Sigma-Aldrich, St. Louis, MO) (V/V) (TBS-T) and 5% (W/V) non-fat milk powder then incubated overnight at 4°C in blocking buffer containing either polyclonal rabbit anti-SARS-CoV-2 Spike antibody (1:3000 dilution) (Sino Biological) or polyclonal rabbit anti-β-actin antibody (1:1000 dilution) (Cell Signaling). Membranes were then incubated for 1 hour at room temperature with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:75, 000 dilution) (ThermoFisher, Ottawa, ON) in blocking buffer and developed using SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher, Ottawa, ON) and a ChemiDoc MP imaging system (Bio-Rad, Saint-Laurent, QC).

Tamming L.A., Duque D., Tran A., Zhang W., Pfeifle A., Laryea E., Wu J., Raman S.N., Gravel C., Russell M.S., Hashem A.M., Alsulaiman R.M., Alhabbab R.Y., Gao J., Safronetz D., Cao J., Wang L., Chen W., Johnston M.J., Sauve S., Rosu-Myles M, & Li X. (2022). DNA Based Vaccine Expressing SARS-CoV-2 Spike-CD40L Fusion Protein Confers Protection Against Challenge in a Syrian Hamster Model. Frontiers in Immunology, 12, 785349.

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