Stain free system

Manufactured by Bio-Rad

The Stain Free System is a laboratory equipment product by Bio-Rad. It is used for the detection and quantification of proteins in gel-based applications.

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6 protocols using stain free system

Quantitative Western Blot Analysis of Muscle Proteins

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Western blot analysis was performed on muscle homogenates as previously described [16 (link)]. Briefly, after electrophoretic separation on a 4–20% gradient acrylamide gel (Stain-free precast gel, Biorad, France) and electrotransfer to Immobilon P (Biorad, France), the membrane was incubated with primary antibodies and then HRP-labelled secondary antibodies (Jackson ImmunoResearch Laboratories). Signal quantification was performed using a ChemiDoc Touch apparatus (Biorad, France) and the Image Lab software (Biorad). The amount of the chosen protein in each sample was corrected for differences in loading using either the amount of myosin, GAPDH or the total amount of proteins using the stain free system from Biorad, and normalized to the amount of the same protein present in the control, set to 100% as described previously [15 (link)]. Ten human controls (muscle biopsy from individuals non-affected by neuromuscular disease) of different age have been used, from 3.5 to 64 years. For each sample (mouse and human), 2–3 Western blots have been performed, and the value for each sample corresponds to the mean ± SEM of the different Western blots.

Pelletier L., Petiot A., Brocard J., Giannesini B., Giovannini D., Sanchez C., Travard L., Chivet M., Beaufils M., Kutchukian C., Bendahan D., Metzger D., Franzini Armstrong C., Romero N.B., Rendu J., Jacquemond V., Fauré J, & Marty I. (2020). In vivo RyR1 reduction in muscle triggers a core-like myopathy. Acta Neuropathologica Communications, 8, 192.

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Cellular Fractionation and Western Blot Analysis

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Total cells lysis was performed using a lysis buffer containing 1.5 mM EDTA, 50 mM Hepes pH 7.4, 150 mM NaCl, 10% (v/v) glycerol and 1% (v/v) NP40. Mitochondria enriched fractions were isolated from cultured cells using a specific buffer containing 10 mM Tris, 1 mM EDTA and 250 mM sucrose as previously described [47 (link)]. Total cellular extracts and mitochondrial fractions were loaded onto a 4–15% SDS-PAGE gel (Bio-Rad), transferred onto nitrocellulose membrane and revealed with different commercial antibodies: β-catenin Cell Signaling (8480S), Claudin-1 Cell Signaling (13255S), EIF3F abcam (ab64177), EIF3F Novus (NBP1-77997), FLAG M2 Novus (NBP1-06712), PGC1α Santa Cruz (sc-13067), SNAIL Cell Signaling (3879S), STAT3 Cell Signaling (8768S), TFAM abcam (ab131607), TOM20 Santa Cruz (sc-11021), ZO-1 Cell Signaling (8193S), and anti-βActin Sigma Aldrich (A1978). Fluorescent secondary antibodies and HRP-coupled secondary antibodies were used for revelation using respectively Odyssey instrument (LI-COR) or ChemiDoc imaging instrument (Bio-Rad). Protein expressions were normalization using standard protein expression or total loading protein (Stain-free system Bio-Rad).

Esteves P., Dard L., Brillac A., Hubert C., Sarlak S., Rousseau B., Dumon E., Izotte J., Bonneu M., Lacombe D., Dupuy J.W., Amoedo N, & Rossignol R. (2019). Nuclear control of lung cancer cells migration, invasion and bioenergetics by eukaryotic translation initiation factor 3F. Oncogene, 39(3), 617-636.

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Quantitative Western Blotting Analysis

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Western blotting studies were performed as previously described^{59 (link)}. Proteins were resolved on 10% Mini-PROTEAN TGX stain-free pre-cast gels (Bio-Rad) and transferred onto PVDF using the Trans-Blot Turbo transfer system. Membranes were probed with specific primary antibodies against proteins of interest overnight at 4 °C diluted in appropriate blocking buffer followed by incubation with a HRP-conjugated secondary. Membranes were developed in SuperSignal West Pico PLUS Chemiluuminescent substrate (#34580, Thermo Scientific) and imaged using ChemiDoc Imaging System (BioRad). Data are presented as fold change in protein expression relative to control groups (D0 animals or vehicle-treated cells) after normalisation to loading controls (β-tubulin). Total protein was used to assess equal loading in studies of different tissues using the stain-free system (BioRad).

Smith E.R., Tan S.J., Holt S.G, & Hewitson T.D. (2017). FGF23 is synthesised locally by renal tubules and activates injury-primed fibroblasts. Scientific Reports, 7, 3345.

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Western Blot Analysis of Melanoma Proteins

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Thirty μg total protein melanoma lysates (MPER, Thermo Fisher – 78501) were separated on 4-15% Bis-Tris gels (Bio-Rad) and transferred to nitrocellulose membranes. The membranes were incubated overnight with antibody (Dilutions - 1:1000 for YAP/TAZ antibody (Cell Signaling Technology – D24E4), 1:1000 ARPC5 (Proteintech - 16717-1-AP), 1:1000 ARPC5L (Proteintech – 22025-1-AP), 1:10000 for GAPDH (Cell Signaling – D16H11)) in nonfat 5% milk (Santa Cruz – sc-2324). The membranes were washed with 1X TBS-T four times for 15 minutes and incubated with 1:4000 anti-rabbit IgG HRP-linked antibody (Cell Signaling – 7074). Membranes were developed with Clarity Western ECL substrate according to the manufacturer’s instructions (Bio-Rad). Blots were normalized with GAPDH or total protein input (Stain Free System, Bio-Rad). All Western analysis shown are representatives of at least three independent experiments.

Lui J.W., Moore S.P., Huang L., Ogomori K., Li Y, & Lang D. (2021). YAP facilitates melanoma migration through regulation of actin-related protein 2/3 complex subunit 5 (ARPC5). Pigment cell & melanoma research, 35(1), 52-65.

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Western Blot Analysis of Transcription Factors

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Cells were lysed in M-PER (Mammalian Protein Extraction Buffer) buffer. Proteins (50 μg) were separated on a 4–15% Bio-Rad stain free gels (10 well 4–15% Biorad-Stain-free-gel cat no 4568084), transferred to a nitrocellulose membrane, and probed with antibodies against MET (Cell Signaling Technology Cat#8198, RRID:AB_10858224), ETS1 (Santa Cruz Cat#sc-55581, RRID:AB_831289), ETS1/2 (for detecting DN-ETS, (Santa Cruz Cat#sc-374509, RRID:AB_10987669), ETV4 (Proteintech Cat#10684–1-AP, RRID:AB_2100984), ETV5 Sigma-Aldrich Cat#WH0002119M2, RRID:AB_1841526). GAPDH (Cell Signaling Cat#5174, RRID:AB_10622025) or total protein input (Stain Free System, Bio-Rad) was used as a loading control. ImageLab (BioRad) was utilized to quantify band intensity that was normalized to loading controls. All western analyses were repeated minimally in triplicate.

Huang L., Zhai Y., La J., Lui J.W., Moore S.P., Little E.C., Xiao S., Haresi A.J., Brem C., Bhawan J, & Lang D. (2021). Targeting pan-ETS factors inhibits melanoma progression. Cancer research, 81(8), 2071-2085.

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Wnt5a and CDC42 Activation Assays

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For Wnt5a expression analysis, Western blotting was performed with lysates from BM primary stroma of Wnt5a^+/+ and Wnt5a^+/− mice containing 10% glycerol, 25 mM sodium fluoride, 1 mM sodium orthovanadate, and a protease inhibitor cocktail (Roche). After blotting the proteins on polyvinylidene difluoride membranes (EMD Millipore), 2% BSA in TBS with 0.1% Tween-20 was used for blocking and dilution of anti-Wnt5a antibody. Primary antibody was detected with an anti–rabbit horseradish peroxidase-conjugated secondary antibody and developed with the use of Super Signal chemiluminescent substrates.
Relative levels of GTP-bound CDC42 were determined by an effector pull-down assay. In brief, lineage-depleted BM cells (10⁶) were lysed in a Mg²⁺ lysis/wash buffer (EMD Millipore), including protease inhibitors as in the previous paragraph. Samples were incubated with PAK-1–binding domain/agarose beads (EMD Millipore), and bound (activated) and unbound (nonactivated) CDC42 fractions were probed by immunoblotting with an anti-CDC42 antibody (rabbit polyclonal; EMD Millipore). Activated protein was normalized to total protein (Stain Free System; Bio-Rad Laboratories) and the relative amount was quantified by densitometry (ChemiDoc Imaging System; Bio-Rad Laboratories).

Schreck C., Istvánffy R., Ziegenhain C., Sippenauer T., Ruf F., Henkel L., Gärtner F., Vieth B., Florian M.C., Mende N., Taubenberger A., Prendergast Á., Wagner A., Pagel C., Grziwok S., Götze K.S., Guck J., Dean D.C., Massberg S., Essers M., Waskow C., Geiger H., Schiemann M., Peschel C., Enard W, & Oostendorp R.A. (2017). Niche WNT5A regulates the actin cytoskeleton during regeneration of hematopoietic stem cells. The Journal of Experimental Medicine, 214(1), 165-181.

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