Protein levels of PLIN5 in human muscle tissue were determined by Western blot in 8/10 individuals from the subgroup. Tissues were lysed using Bio‐Plex Cell Lysis kit (171‐304011; Bio‐Rad) and equal amounts of protein were loaded onto the gels (Figure S1 ). After gel electrophoresis, proteins were transferred by Western blotting and a Revert total protein stain (LI‐COR Bioscience, Westburg) was performed to determine the protein quality and the protein quantity on the blots. Then, the membranes were blocked with LI‐COR Blocking buffer for 30 min and incubated with a primary antibody against PLIN5 (1:2500, diluted in LI‐COR Blocking buffer) overnight at room temperature. A IRDye800‐conjugated secondary antibody was used for visualization PLIN5 by an Odyssey near infrared scanner (LI‐COR Biosciences). Western blots were quantified with Image Studio version 5.2 (LI‐COR Biosciences) and values were normalized to total protein stain.
Total protein stain
Manufactured by LI COR
The Total Protein Stain is a laboratory product designed for the detection and visualization of proteins in polyacrylamide gels. It provides a sensitive and reliable method for staining total proteins, allowing for the quantification and analysis of protein samples.
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Lab products found in correlation
Odyssey clx imaging system, by LI COR (2 mentions)
Odyssey near infrared scanner, by LI COR (1 mentions)
Revert total protein stain, by LI COR (1 mentions)
Bio plex cell lysis kit, by Bio-Rad (1 mentions)
Image studio version 5, by LI COR (1 mentions)
Tris glycine sds buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey application software 3, by LI COR (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Adi spa 801, by Enzo Life Sciences (1 mentions)
Mops running buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey xf imager, by LI COR (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Irdye 680rd goat anti mouse, by LI COR (1 mentions)
H3k4me2, by Active Motif (1 mentions)
H3k4me3, by Active Motif (1 mentions)
Odyssey clx scanner, by LI COR (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Image studio, by LI COR (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
Irdye 680rd goat anti mouse igg secondary antibody, by LI COR (1 mentions)
11 protocols using total protein stain
1
Quantifying PLIN5 Protein Levels in Human Muscle
2
Western Blot Analysis of Protein Signaling
3
Whole Worm Lysate Western Blot
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Standard Western blotting methods were used. Twenty micrograms of total worm lysate from disruption of 800–1 000 whole worms described above were boiled in Laemmli buffer and loaded onto a gradient SDS-PAGE gel, separated, and transferred onto PVDF membrane. Membranes were stained with LI-COR Total Protein Stain and imaged using the LI-COR Odyssey. Then, membranes were probed with anti-HspB1 antibody (ADI-SPA-801, ENZO Life Sciences, Farmingdale, NY;1:1 000 overnight at 4°C), followed by a fluorescent secondary antibody and imaging on the LI-COR Odyssey and analysis on LI-COR Imaging software.
4
Western Blot Protein Analysis
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Cells were washed one time with PBS before pelleting. Pellets were resuspended in 1x RIPA buffer with protease and phosphatase inhibitors added. After shaking samples at 4°C at 600 rpm, samples were centrifuged for 10 minutes at 14,000xg. The lysates were then quantified using Bradford assay and diluted to 1x LDS. Samples were loaded on a 4–12% Bis-Tris gel (ThermoFisher, NP0321BOX) and run in MOPS Running Buffer (ThermoFisher, NP0001) followed by transfer to PVDF membranes using a TransBlot Turbo Transfer System at 25 V, 1.3 A, for 12 min (Bio-Rad). Total protein was quantified using total protein stain (LI-COR, 926–11011) and blocked in 5% milk in 1x TBST. Blot was incubated overnight with primary antibody in 1x TBST with 5% BSA at 4°C. Blots were then washed in 1x TBST, incubated in secondary antibody for 1 hour at room temperature in 5% milk, and washed three times for 10 minutes. Blots were incubated with HRP substrate and imaged on the LI-COR Odyssey XF Imager.
5
Drp1 Protein Expression in Ischemic Brain
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Protein extract (30 µg) from the ischemic hemisphere was loaded into 4–12% Novex gels and run at 60 V for 30 min and then at 100 V for 90 min. The protein was then transferred onto a PDVF membrane and probed with an antibody for Drp1 (anti-mouse, Abcam, 1:1000 µl) and conjugated to a fluorescent secondary antibody (LiCOR goat anti-mouse IRDye 680RD), normalizing to total protein (LiCOR Total Protein Stain).
6
Lysate Preparation and Immunoblotting Protocol
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Lysates for immunoblotting were prepared using either 0.2 M NaOH treatment or bead-beating in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) (Jezek et al., 2017a (link); Tran et al., 2018 (link)). Protein concentrations were determined by Bradford assay and normalized to load equivalent amounts on SDS–PAGE. Proteins were transferred to Immobilon-FL polyvinylidene difluoride membrane (Fisher Scientific). Blots were probed overnight with primary antibodies, followed by incubation with the appropriate secondary antibody. Blots were imaged using an Odyssey CLx Scanner and processed using ImageStudio (LiCor). Total Protein Stain (LiCor) was used to determine total protein amounts for quantitation. Antibodies used in this study were c-MYC 9E10 (Novus Biologicals NB600-302SS), monoclonal anti-FLAG M2 (Sigma-Aldrich F1804), H3K4me3 (Active Motif 39159), H3K4me2 (Active Motif 39142), H3 (Abcam ab1791), H4 (Abcam ab31830), IRDye 680RD goat anti-mouse immunoglobulin G (IgG) (LiCor 926-68070), and IRDye 800CW goat anti-rabbit IgG (LiCor 926-32211).
7
Whole-cell Protein Extraction and Western Analysis
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Cell pellets were resuspended using lysis buffer (25 mM HEPES, pH 7.4, 0.4 M KCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 1% NP-40, 1 mM PMSF, and 2 μg/mL of leupeptin) of 2.5× volume of cell pellets. After three cycles of freeze/thaw, lysates were kept on ice for 30 min and were then centrifuged at 16,000 g for 10 min at 4 °C. The supernatants were defined as whole-cell lysates and were subjected to BCA assay and LI-COR Western analysis. The protocol for Western analysis was described previously [48 (link)] with minor modification. Fifteen percent acrylamide gels were transferred for 3 h at 4 °C. After the wet transfer, Total Protein Staining was performed using LI-COR Total Protein Stain. Membranes for the examination of LC3B levels for autophagic flux were dried overnight and wet with PBS before blocking. The transferred nitrocellulose membranes were blocked in PBS with 5% BSA for 1 h. Dilutions for antibodies were as follows: 1:1,000 for p23 (JJ3), p62, LC3B and AHR (A-3x); 1:2,000 for AHR (SA210); 1:5,000 for β-actin; 1:200 for HSP90 (N-17). If not specified, Western bands were normalized using Total Protein Stain. Results were obtained and analyzed using an LI-COR Odyssey CLx imaging system.
8
Western Blot Analysis of Viral Proteins
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Lysate from cell pellets containing 3E6 cells was mixed with loading dye (VWR Radnor, PA) and denatured before being equally loaded onto a precast gel (Bio-Rad Hercules, CA) with a protein ladder (LI-COR BioSciences, Lincoln, NE). The gel was transferred onto a nitrocellulose membrane using the iBLOT transfer system (Thermo Fisher, Waltham, MA). A total protein stain (LI-COR BioSciences) was applied, and the membrane was imaged on the Odyssey CLx Imaging System (LI-COR BioSciences) for quantification purposes. The membrane was incubated in Odyssey PBS blocking buffer (LI-COR BioSciences) prior to incubation with primary antibody overnight at 4°C. B1 primary antibody (American Research Product, Waltham MA) was used to detect VP1, VP2, and VP3 expression at a 1:1,000 dilution, and anti-Rep (American Research Product, Waltham MA) was used at a 1:250 dilution in blocking buffer. After washing, the membrane was incubated in the secondary antibody, IRDye 680RD Goat anti-Mouse IgG Secondary Antibody (LI-COR biosciences), before being imaged on the Odyssey CLx. For quantification, the signal from each band was measured and normalized against the total protein stain signal for each lane.
9
Burmese Python Liver Protein Analysis
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Burmese python liver was homogenized in radioimmunoprecipitation assay buffer (#9806; Cell Signaling Technology), supplemented with protease and phosphatase inhibitors cocktail (#78442; Thermo Fisher Scientific), and centrifuged at 14,000 g for 15 min. 10 μg of protein (python) from the supernatant was resolved by SDS-PAGE and analyzed by Western blot using antibodies from Cell Signaling Technology: p-pASK1 (Thr845; #3765), p-MKK3 (Ser198)/p-MKK6 (Ser207; #12280), p-p38 (Thr180/Tyr182; #4511), p-AKT (Ser473; #4058), and FASN (#3180). Total protein stain (LI-COR #926-11016; Lincoln) was used for Western blot normalization (Figs. S4, C–H; and S5 ).
10
Western Blot Analysis of LAMP1 and EEA1
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Eight micrograms of each sample were electrophoresed through a 15-well, 4–20% SDS–PAGE gel (Bio-Rad #4561096) at 100 V for 1 hr at RT and transferred onto a nitrocellulose membrane (GE Life Sciences #GE10600002) at 100 V for 70 min at RT on ice. Membranes were incubated with Total Protein Stain for 5 min at RT (LI-COR # 926–11015), rinsed, and imaged at 700 nm using an Odyssey Fc imaging system (LI-COR) to determine protein loading. Membranes were then briefly incubated with REVERT solution and blocked with Odyssey blocking buffer (LI-COR #927–50000) for 1 hr at RT. Membranes were probed with one primary antibody at a time for 24 hr at 4°C and then washed four times with 1× TBST before incubating with secondary antibody at RT for 1 hr. Membranes were washed four times with TBST and then imaged with an Odyssey Fc imaging system at 700 nm and 800 nm. Order of probes: LAMP1 then EEA1. We determined the optical density of the bands using Odyssey Fc Image Studio software. Data obtained from three independent experiments and statistically analyzed using GraphPad Prism (version 8) software.
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