Cells were lysed on ice for 30 min in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton-X 100, 2 mM EDTA, 0.2% SDS, and complete protease inhibitor (Roche). Protein lysates were quantified with Bradford analysis (Thermo Fisher Scientific), and 10 µg of each lysate was separated through SDS-PAGE gel electrophoresis (Invitrogen). Protein was then transferred onto Immobilon Transfer Membranes (EMD Millipore) through the fast semi-dry transfer method (Thermo Fisher Scientific). Membranes were stained with anti–Munc13-4, anti–Munc18-2, anti-syntaxin 11 (Proteintech), anti-STAT4, anti-pSTAT4 (Abcam), anti-BRG1 (Abcam), anti-ELF1 (Santa Cruz Biotechnology, Inc.), or anti–β-actin (Sigma-Aldrich) primary antibodies. Secondary staining was performed with anti–rabbit or anti–mouse antibodies conjugated to horseradish peroxidase (Invitrogen). Chemiluminescence was visualized with an ImageQuant LAS 4000 Mini (GE Healthcare), and densitometry values were quantified with ImageJ software (National Institutes of Health).
Sds page gel electrophoresis
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
SDS-PAGE gel electrophoresis is a laboratory technique used to separate and analyze proteins based on their molecular weight. It utilizes an electric field to move charged proteins through a polyacrylamide gel matrix, allowing them to be separated and visualized.
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Lab products found in correlation
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Anti stat4, by Abcam (1 mentions)
Anti elf1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Immobilon transfer membrane, by Merck Group (1 mentions)
Anti brg1, by Abcam (1 mentions)
Las 4000, by Cytiva (1 mentions)
Superose 6 increase 10 300 gl column, by GE Healthcare (1 mentions)
3 protocols using sds page gel electrophoresis
1
Western Blot Analysis of Cell Signaling
2
Recombinant Protein Purification Workflow
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Recombinant pETDuet plasmids were transformed into SoluBL21 E. coli (Genlantis, cat. C700200). E. coli were grown in M9 minimal media (according to manufacturer's protocol) at 16C, and protein expression was induced by IPTG. Unless otherwise stated, all subsequent steps were done at 4 C. Cells were spun down and lysed prior to protein isolation by standard His Nickel-NTA (Invitrogen, cat. R90101) column chromatography. The Nickel-NTA isolated fraction was further purified using Superose 6 Increase column 10/300 GL (GE Healthcare) size-exclusion chromatography. The choice of column provided the appropriate fractionation range and resolution such that the expected protein tetramer could be purified from smaller incomplete or degraded products. Subsequent analysis of column fractions was performed by visual inspection of SDS-Page gel electrophoresis (Invitrogen, cat. NP0322) and fractions meeting expected size and purity were pooled and concentrated (50 mM TrisHCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% v/v Glycerol, pH = 7.3 at 25 °C).
3
Recombinant UFSP2 Protease Assay
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A previously described expression construct for mouse UFSP2 (NM_138668.2) was modified to incorporate the heterologous BHD mutation (c.844T>C, p.Tyr282His). [7] (link) The wild-type (WT) (UFSP2 WT) and mutant (UFSP2 BHD) constructs together with a GST-Ufm1-HA construct were expressed in Escherichia coli and purified. Purified proteases were incubated with GST-Ufm1-HA at 37 o C for 1 hour and the products were separated by SDS-PAGE gel electrophoresis (Invitrogen Ltd., UK). Protein bands were visualised by staining with Coomassie blue R-250.
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