Sds bis tris gel

Manufactured by Thermo Fisher Scientific

The SDS Bis-Tris gel is a type of polyacrylamide gel used for electrophoretic separation of proteins. It is designed to maintain a neutral pH during the separation process. The gel is composed of bis-acrylamide and Tris-HCl buffer system, which allows for the separation of proteins based on their molecular weight.

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Lab products found in correlation

Trypsin gold, by Promega (3 mentions) Colloidal blue, by Thermo Fisher Scientific (2 mentions) Lds page buffer, by Thermo Fisher Scientific (2 mentions) Tissue homogenizer, by Qiagen (2 mentions) α tubulin, by Merck Group (1 mentions) Cellulose acetate membrane, by Bio-Rad (1 mentions) Anti gfp antibody, by Roche (1 mentions) Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions) Ndufs3, by Abcam (1 mentions) Mthsp70, by Abcam (1 mentions) Hrp conjugated goat anti rabbit antibody, by Agilent Technologies (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Bicinchoninic acid bca assay, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Bis-Tris gels (1248 citations) Novex Bis-Tris SDS-acrylamide gels (15 citations) Bis-Tris SDS-polyacrylamide gel (11 citations) SDS PAGE 4–12% Bis-Tris gels (7 citations) Bolt SDS-PAGE 4%–12% Bis-Tris gels (6 citations) 4-to-12% bis-Tris SDS-PAGE gels (4 citations) SDS NuPAGE Bis-Tris 4–12% gels (4 citations) SDS–4 to 12% bis-Tris protein gels (4 citations) NuPAGE Bis-Tris gradient SDS-PAGE gels (3 citations) Novex Bis-Tris 4–12% SDS−PAGE gel (3 citations)

7 protocols using sds bis tris gel

Detecting Protein Aggregates via Slot Blot and Western Blot

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100 μg of protein samples were applied on to cellulose acetate membrane with 0.22 μm pore size (Schlechtes + Schule), assembled in vacuum slot blotter (Bio-Dot, Bio-Rad). Membrane was washed with 0.2% SDS five times on the blotter and subjected to antibody incubation for detecting aggregated protein retained on the membrane. Briefly, membrane was incubated with anti-GFP antibody (1:3000 dilution in 5% milk in PBS, Roche) overnight in a cold room. Membrane was washed with PBST (PBST with 0.05% Tween-20) for three times, then incubated with secondary antibody (donkey anti-mouse antibody conjugated with HRP, 1:5000 dilution in 5% milk in PBS, Jackson Immuno Research). Membranes were washed with PBST three times and exposed to film after applying ECL solutions (Pierce) to visualize the protein bands.
For SDS-PAGE, 20-40 μg of protein samples were loaded on 4%-12% bis-tris SDS gel (Invitrogen). The gel was transferred to nitrocellulose membrane (GE) using XCell II blot module (Invitrogen). Then, membranes were incubated with antibodies and exposed to film as described above. NDUFS3 (Abcam), mtHSP70 (Abcam), α-Tubulin (Sigma), GFP (Roche), and cytosolic HSP70 (HSP70/HSP72, Enzo life), HSP60 antibody supernatant (University of Iowa) were used to probe the membrane.
The Western blot bands intensity was measured using ImageJ software.

Kim H.E., Grant A.R., Simic M.S., Kohnz R.A., Nomura D.K., Durieux J., Riera C.E., Sanchez M., Kapernick E., Wolff S, & Dillin A. (2016). Lipid Biosynthesis Coordinates a Mitochondrial-to-Cytosolic Stress Response. Cell, 166(6), 1539-1552.e16.

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Western Blot Analysis of VAR2CSA Epitopes

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Western blot was performed to determine if the individual Nbs recognized continuous or discontinuous VAR2CSA epitopes. Recombinant VAR2CSA protein (0,4 µg/lane) was loaded on an 8–12% Bis-Tris SDS gel (invitrogen) under reduced or non-reduced conditions. The separated proteins were transferred to nitrocellulose membranes by wet blotting. After blocking non-specific sites with 5% skim milk in TBS+0.05% Tween 20 (TBST), the membranes were incubated for 1½ hour at RT sequentially with VAR2CSA-specific nanobody (diluted to 50 nM in TBST), rabbit polyclonal anti-camel Ab (diluted 1∶2000 in TBST) and HRP-conjugated goat anti-rabbit antibody (DAKO; P0448 diluted 1∶3000 in TBST). Between incubations, the membranes were washed three times with TBST and Nb recognition of VAR2CSA was detected by chemiluminescence.

Ditlev S.B., Florea R., Nielsen M.A., Theander T.G., Magez S., Boeuf P, & Salanti A. (2014). Utilizing Nanobody Technology to Target Non-Immunodominant Domains of VAR2CSA. PLoS ONE, 9(1), e84981.

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Oxylipin-Induced Proteomic Changes in P. aeruginosa

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P. aeruginosa PAO1 and ΔodsR strains were grown in quadruple in M63 complete medium (3 mL) up to an OD600 = 1 at 30 °C and shaking (240 rpm). Each strain was then treated with 0.1 mg mL⁻¹ of OA, 10-HOME or 7,10-DiHOME oxylipin or kept untreated as baseline control and incubated for additional 2 h at 30 °C and shaking (240 rpm). Bacterial cells were collected by centrifugation (10,000 rpm, 5 min) from 1 mL aliquots of each culture and frozen at −80 °C until use. Bacterial pellets were then thawed and lysed with 20 µL of NuPAGE LDS sample buffer, reduced, denatured and separated by one-dimensional polyacrylamide gel electrophoresis (1D PAGE) as previously referenced^{30 (link)}. This step was carried out on an SDS Bis-Tris gel (10%, Invitrogen) as per the manufacturer’s instructions. The gels were stained, the entire lane for each sample were partitioned into 3 MW fractions and each gel plug was equilibrated in 100 mM ammonium bicarbonate (AmBc). Each gel plug was then digested with Trypsin Gold (Promega) following the manufacturer’s instruction, and peptide extracts were reconstituted in 0.1% Formic Acid/ddH₂O at ~0.1 µg µL⁻¹. Mass spectrometry runs were carried out, and the data processed, searched, filtered, grouped and quantified as previously reported in detail (Ludwig et al.^{31 (link)}; under section 2.5 nLC-ESI-MS2 and Protein IDs for GeLC)^{31 (link)}.

Martínez E., Cosnahan R.K., Wu M., Gadila S.K., Quick E.B., Mobley J.A, & Campos-Gómez J. (2019). Oxylipins mediate cell-to-cell communication in Pseudomonas aeruginosa. Communications Biology, 2, 66.

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Protein Extraction and Fractionation Protocol

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Each cell pellet was lysed in B-per supplemented with lysozyme, Dnase I, and EDTA using the B-PER Kit (Pierce, Thermo Fisher Scientific) following manufacturers’ instructions. Protein concentrations of the cell lysates were determined with the BCA protein assay (Pierce, Thermo Fisher Scientific). Twenty micrograms (20 µg) of protein from each sample was diluted in LDS PAGE buffer (Invitrogen) followed by reducing, heat denaturing, and separation on a 10% SDS Bis-Tris gel (Invitrogen). The gel was stained overnight with Colloidal Blue (Invitrogen), and the two most abundant bands, A and B (Figure 1), were first carefully excised. Based on staining intensities, the rest of the gel lane was then cut into six nearly equal fractions from the top to bottom, and all eight of the resultant gel bands were then equilibrated in 100 mM ammonium bicarbonate (AmBc). Gel slices were reduced, carbidomethylated, dehydrated, and digested with Trypsin Gold (Promega) as per manufacturers’ instructions. Following digestion, peptides were extracted, volumes were reduced in a SpeedVac to near dryness, and re-suspended to 20 µL using 95% ddH₂O/5% ACN/0.1% formic acid (FA) prior to analysis by 1D reverse phase LC-ESI-MS2 (as outlined below).

Ellis M.E., Mobley J.A., Holmes R.P, & Knight J. (2016). Proteome Dynamics of the Specialist Oxalate Degrader Oxalobacter formigenes. Journal of proteomics & bioinformatics, 9(1), 19-24.

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Protein Extraction from HFD-fed Mouse Livers

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Proteins were extracted from the livers of HFD-fed WT male mice 6–8 weeks of age. Livers were homogenized using a tissue homogenizer (Qiagen) in ice-cold buffer (20 mm Tris (pH 7.5), 2.7 mm KCl, 1 mm MgCl₂, 1% Triton X-100, 10% (w/v) glycerol, 1 mm EDTA, and 1 mm DTT) supplemented with protease (Thermo Scientific) and phosphatase inhibitor mixture (Millipore). Samples were then centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatants were collected. The protein content of the supernatant was determined using a BCA assay (Thermo Scientific). 50 μg of each sample was loaded for Western blotting. Proteins were resolved on a 4% to 12% gradient SDS BisTris gel (Invitrogen). The IMP2 antibody is described in Ref. 4 (link), and sources of other antibodies are shown in Table S1. All commercial antibodies were used at 1:1000 dilution, with specificity validated by the vendors.

Regué L., Minichiello L., Avruch J, & Dai N. (2019). Liver-specific deletion of IGF2 mRNA binding protein-2/IMP2 reduces hepatic fatty acid oxidation and increases hepatic triglyceride accumulation. The Journal of Biological Chemistry, 294(31), 11944-11951.

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Protein Extraction and Fractionation Protocol

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Ellis M.E., Mobley J.A., Holmes R.P, & Knight J. (2016). Proteome Dynamics of the Specialist Oxalate Degrader Oxalobacter formigenes. Journal of proteomics & bioinformatics, 9(1), 19-24.

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Protein Extraction and Analysis from Islet Cells

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Proteins were extracted using a tissue homogenizer (Qiagen). Purified islets from 8-week-old, HFD-fed mice were homogenized in ice-cold buffer (20 mM Tris [pH 7.5], 2.7 mM KCl, 1 mM MgCl₂, 1% Triton X-100, 10% [wt/vol] glycerol, 1 mM EDTA, 1 mM dithiothreitol) supplemented with protease (Thermo Scientific) and a phosphatase inhibitor cocktails (Millipore). Samples were then centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatants were collected. The protein content of the supernatant was determined using a bicinchoninic acid (BCA) assay (Thermo Scientific). Aliquots of each extract containing 50 μg protein were loaded for Western blotting. Proteins were resolved on a 4–12% gradient SDS Bis-Tris gel (Invitrogen, Carlsbad, CA). Antibodies for immunoblotting are listed in Supplementary Table 2. For protein half-life assay, 250 mM cycloheximide was used to inhibit mRNA translation; protein half-life was estimated from immunoblot of extracts prepared before and at various times after cycloheximide addition.

Regué L., Zhao L., Ji F., Wang H., Avruch J, & Dai N. (2021). RNA m6A reader IMP2/IGF2BP2 promotes pancreatic β-cell proliferation and insulin secretion by enhancing PDX1 expression. Molecular Metabolism, 48, 101209.

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