Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) for both Sup35 and Rnq1 proteins was performed as described previously with generated polyclonal αSup35 (kind gift of S. Lindquist) and αRnq1 antibodies, respectively (Huang et al., 2013 (link); Dulle et al., 2014 (link)). The solubility assay of Rnq1 was performed as described (Bardill and True, 2009 (link)). Briefly, cell lysates in detergent-containing lysis buffer were subjected to ultracentrifugation at 80,000 rpm for 30 min at 4°C to separate into soluble and insoluble fractions that were analyzed by SDS-PAGE and western blot with a polyclonal αRnq1 antibody. Well-trap assays were performed as described (Stein and True, 2014 ). Additional antibodies used to analyze protein expression included: two different αSis1 antibodies (here referred to as αSis1.1 (COP-COP-080051, Cosmo Bio Co.) and αSis1.2 (a kind gift from E. Craig)), αHdj1 (ADI-SPA-400, Enzo Life Sciences), and αPgk1 (A6457, Molecular Probes). All results are representative of at least three independent experiments, each using at least five yeast transformants.
Adi spa 400
Manufactured by Enzo Life Sciences
The ADI-SPA-400 is a laboratory instrument designed for spectrophotometric analysis. It is capable of performing absorbance measurements across a range of wavelengths, providing users with data on the optical properties of samples.
Automatically generated - may contain errors
Lab products found in correlation
A6457, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab93806, by Abcam (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Ab32072, by Abcam (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Adi spa 800, by Enzo Life Sciences (1 mentions)
4 protocols using adi spa 400
1
Analyzing Yeast Prion Protein Aggregation
2
Western Blot Analysis of Protein Expression
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Cells were washed once with phosphate buffered saline (PBS) and lysed with radioimmunoprecipitation assay buffer (RIPA buffer) supplemented with protease and phosphatase inhibitor cocktails (Roche). Clarified cell extracts were mixed with equal volume of 2× SDS loading buffer and boiled at 95°C for 5 min. Proteins were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was blocked with 5% non-fat milk in PBST buffer (1x PBS, 0.05% Tween 20) followed by incubating with primary and secondary antibodies diluted in blocking buffer. The blots were developed with ECL (enhanced chemiluminescence) and exposed to X-ray film. The primary antibodies for PKR (12297S), eIF2α(5324S), phosphorylated eIF2α (Ser51)(9721S) and P58IPK (2940S) were from Cell Signaling Technologies. The primary antibody for phosphorylated PKR was from Abcam. The primary antibodies for FLAG tag (F1804), β-actin (A5441) and GFP(G1546) were from Sigma. The primary antibody for c-Myc tag, RPS19(sc-100836) were from Santa Cruz. The primary antibody for Hsp40 (ADI-SPA-400) was from Enzo Life Science. The primary antibody for RPL4(11302-1-AP) was from proteintech. The polyclonal antibody for ANDV and SNV NP was produced in our lab. The antibody for CCHFV NP (ab190657) was from abcam.
3
Western Blot Analysis of Cellular Proteins
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Tissues or cells were lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors. Protein lysates were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidine difluoride membranes. Proteins were detected by standard Western blotting procedures. Antibodies were diluted 1:1000 for BMAL1 (Abcam, ab93806), CRY1 and CRY2 (98 (link)), c-MYC (Abcam, ab32072), HSF1 (CST-12972), KRASG12D (CST-14429), p-ERK1/2 (CST-4370), and ERK1/2 (CST-4695); 1:2000 for REV-ERBα (33 ), DNAJB1 (Enzo, ADI-SPA-400), and HSP90AA1 (GTX109753); 1:10,000 for LAMIN A (Sigma-Aldrich, L1293); 1:50,000 for ACTIN (Sigma-Aldrich, A1978). Imaging and band quantification were carried out using ChemiDoc XRS+ System (Bio-Rad).
4
Comprehensive Protein Extraction and Western Blot Analysis
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Cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 1% NP40, 250 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 1 mM protease inhibitor (PMSF, aprotinin, leupeptin) and 1 mM DTT. Whole cell lysates were subjected to protein quantification and analyzed by Western blotting. Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.
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