Equal volumes of serum from randomly chosen samples were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 V for 30 min. The membranes were blocked with PBS containing 5% nonfat dry milk and probed separately with each of the antibodies: anti-CKM (mouse monoclonal, 1:1,000, Santa Cruz, sc-365046), anti-COMP (mouse monoclonal, 1:1,000, Santa Cruz, sc374660), anti-EFEMP1 (mouse monoclonal, 1:1,000, Santa Cruz, sc-33722, 1:1,000), anti-FBLN1 (mouse monoclonal, 1:1,000, Santa Cruz, sc25281), anti-GSN (mouse monoclonal, 1:1,000, Abcam, ab11081), anti-PROS1 (rabbit monoclonal, 1:1,000, Santa Cruz, sc-52720). The membranes were washed three times in PBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for the housekeeper protein transferrin. The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45s).
Criterion stain free 4 20 sds page gels
Manufactured by Bio-Rad
The Criterion Stain-free 4–20% SDS-PAGE gels are precast polyacrylamide gels used for electrophoretic separation of proteins. The gels are compatible with stain-free detection technology, which allows for visualization of proteins without the need for traditional staining methods.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc system, by Bio-Rad (9 mentions)
Image lab, by Bio-Rad (8 mentions)
Nih imagej software, by Bio-Rad (3 mentions)
β actin, by Abcam (3 mentions)
α synuclein antibody, by BD (2 mentions)
Nfl antibody, by Cell Signaling Technology (2 mentions)
Anti gsn, by Abcam (1 mentions)
Anti ckm, by Santa Cruz Biotechnology (1 mentions)
Anti c3 antibody, by Santa Cruz Biotechnology (1 mentions)
Tdp 43 antibody, by Proteintech (1 mentions)
Kmb3441, by Thermo Fisher Scientific (1 mentions)
Kmb3481, by Thermo Fisher Scientific (1 mentions)
Presenilin 1, by Santa Cruz Biotechnology (1 mentions)
Anti α synuclein, by BD (1 mentions)
Amersham ecl plus western blot detection system, by GE Healthcare (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Synaptophysin, by Thermo Fisher Scientific (1 mentions)
α synuclein, by BD (1 mentions)
10 protocols using criterion stain free 4 20 sds page gels
1
Western Blot Analysis of Serum Proteins
2
Quantitative Analysis of Neuronal Proteins
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Protein lysates (10 µg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 V for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed with α-synuclein antibody (1:1000, BD Biosciences, 610,787), NFL antibody (1:2000, Cell Signaling, 2835S) or acrolein antibody (1:1000, NOVUS, NB200-556) overnight at 4 °C. They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for housekeeper proteins tubulin or GAPDH. The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45 s). ELISA of α-synuclein (Legend Max Cat no: 844101) was carried out following the manufacturer’s protocol. ELISA of neurofilament light (Novus Cat no: NBP2-81,184) was carried out following the manufacturer’s protocol.
3
Western Blot Analysis of C3 and Acrolein
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Serum (equal volumes) or protein lysates (10 µg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris-HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 volts for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed with anti-C3 antibody (Santa Cruz, sc-28294, 1:1,000) or anti-acrolein antibody (NOVUS, NB200–556, 1:1,000) overnight at 4 °C. They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for housekeeper proteins transferrin (serum) or β-actin (tissue lysate). The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45 s).
4
Western Blotting Procedure for TDP-43 Detection
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Western blotting was carried out as previously described (Phan et al., 2020 (link)). Protein lysates (10 μg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 volts for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed overnight at 4°C with TDP-43 antibody (Proteintech, 10,782-2-AP, 1:2,000) and β-actin (Abcam, ab6276, 1:10,000). They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad).
5
Quantifying Alzheimer's Protein Biomarkers
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Protein extracts (10 µg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS–PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 V for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed overnight at 4°C with the following antibodies: Amyloid Precursor Protein (Sigma-Aldrich, A8717, 1:1000), Presenilin 1 (Santa Cruz, sc-365450, 1:1000) and β-actin (Abcam, ab6276, 1:10 000). They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45s). ELISA of amyloid-β40 (KMB3481, Thermo Fisher Scientific Australia) and amyloid-β42 (KMB3441, Thermo Fisher Scientific Australia) were carried out following the manufacturer’s protocol.
6
Western Blot Analysis of CDH4 and α-Synuclein
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Protein lysates (10μg for tissue, 20μg for cells) were heated at 95°C for 5 min in sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris-HCl, pH 6.8, 8% 2-mercaptoethanol) and then electrophoresed on Criterion stain-free 4-20% SDS-PAGE gels (Bio-Rad). The gels were transferred onto nitrocellulose membranes at 90 volts for 90 min. The membranes were blocked with TBS containing 5% w/v non-fat dry milk and probed with anti-CDH4 antibody (Abcam, ab109242, 1 : 2,000) or anti-α-synuclein (BD Biosciences, #610787, 1 : 2,000) overnight at 4°C. They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for housekeeper protein β-actin. The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45 s).
7
Western Blot Analysis of α-Synuclein and NfL
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Protein lysates (15 µg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris-HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4-20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 volts for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed with α-synuclein antibody (1:1000, BD Biosciences, 610787) or NfL antibody (1:2000, Cell Signaling, 2835 S) overnight at 4 °C. They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for housekeeper proteins GAPDH or β-actin. The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45 s). All blots were derived from the same experiment and that they were processed in parallel. ELISA of plasma α-synuclein (Legend Max #844101) was carried out following the manufacturer’s protocol.
8
Quantitative Western Blot Analysis of LOXL3 Protein
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Serum (equal volumes) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris-HCl, pH 6.8, 8% 2-mercaptoethanol), electrophoresed on Criterion Stain-free 4-20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 volts for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed with anti-LOXL3 antibody (mouse monoclonal, 1:1000, Santa Cruz, sc377216) overnight at 4°C. The membranes were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Protein bands were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for housekeeper proteins transferrin. The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45s).
9
Western Blot Protein Quantification
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Equal concentrations of TBS fraction of the protein extracts (15 μg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol w/v) and separated on Bio-Rad Criterion Stain-free 4–20% SDS-PAGE gels. The gels were activated for 1 min using Bio-Rad chemiDoc MP imaging system prior to transfer of proteins to a 0.45-μm PVDF membrane. The membranes were imaged for total protein using Bio-Rad chemiDoc MP imaging system. Subsequently, the membranes were blocked with 5% milk powder in TBST for 1 h at room temperature and incubated overnight in apoD primary antibody (Santacruz, sc-373965, 1:2000) prior to protein detection using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) with enhanced chemiluminescence (Amersham ECL Plus Western Blot Detection System, GE Healthcare). The protein band in each gel lane was normalized to total protein using Bio-Rad image lab software.
10
Quantitative Western Blot Analysis of Neuronal Markers
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Western blotting was carried out as previously described1 (link). Protein lysates (10 µg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 V for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed overnight at 4 °C with the following antibodies: ELOVL4 (Abcam, ab14925, 1:800), synaptophysin (Invitrogen, MA1-213, 1:1000), RBFOX3 (BioLegend, 834501, 1:2000), NFL (Cell Signaling, 2835S, 1:2000), α-synuclein (BD, 610787, 1:1000), and β-actin (Abcam, ab6276, 1:10,000). They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45 s). ELOVL4 ELISA was carried out following the manufacturer’s protocol (Abbexa, abx387131).
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