SDS–PAGE was performed following the procedure described by Liu and Xiong (2000 ). SDS–PAGE was operated using a vertical slab gel of 1.0 mm thickness with a 4% stacking gel and a 12% resolving gel in a Bio‐Rad mini‐protein electrophoresis system (Bio‐Rad Laboratories, Hercules, CA, USA). An SPI solution (2%, w/v) was centrifuged at 10,000g for 15 min. The supernatant was diluted to 2 mg/ml with distilled water; 30 μL of the diluted supernatant was mixed with an equal volume of sample buffer with and without 5% (v/v) β‐mercaptoethanol (βME). All mixtures were boiled for 3 min before SDS–PAGE.
Mini protein electrophoresis system
Manufactured by Bio-Rad
Sourced in United States, China, Canada
The Mini Protein Electrophoresis System is a compact and versatile instrument designed for the separation and analysis of proteins using polyacrylamide gel electrophoresis (PAGE) technology. The system allows for the efficient separation of protein samples based on their molecular weight, providing researchers with a reliable tool for various applications in biochemistry, molecular biology, and proteomics.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (2 mentions)
Mini trans blot electrophoretic transfer cell, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Gs 800, by Bio-Rad (1 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Surepage, by GenScript (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (1 mentions)
Model 550 microplate reader, by Bio-Rad (1 mentions)
12 protocols using mini protein electrophoresis system
1
SDS-PAGE for Protein Characterization
2
Western Blot Analysis of Spermatocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified spermatocytes from WT and ClpP/ClpX cKO male C57BL/6 mice testes were suspended in lysis buffer [50 mM HEPES-KOH (pH 7.5), 100 mM KCl, 2 mM EDTA, 10% glycerol, 0.1% NP-40, 10 mM NaF, 0.25 mM Na3VO4, and 50 mM β-glycerophosphate] supplemented with complete protease inhibitor (Cat. 04693116001, Roche, Basel, Switzerland). The samples were homogenized and centrifuged at 20,000 g for 20 min at 4 °C, after which the supernatant was retained for western blotting analysis. The proteins in each sample were separated using 4–12% Bis-Tris gels (Cat. M00652, SurePAGE™, GenScript, Nanjing, China) and a mini protein electrophoresis system (Cat. 1658034, BIO-RAD, CA, USA) following the manufacturer’s instructions. The protein bands were transferred to polyvinylidene fluoride (PVDF) membranes (Cat. IPVH00010, Immobilon, Millipore, MA, USA) via a Mini Trans-Blot Electrophoretic Transfer Cell (Cat. 1703930, BIO-RAD, CA, USA). The immunoreactive bands were detected and analyzed with a BIO-RAD ChemiDoc MP imaging System (Cat. 12003154, BIO-RAD, CA, USA) in conjunction with the Image Lab Software (BIO-RAD, CA, USA). The semi-quantitively analysis of relative protein levels in each sample were normalized to β-tubulin or β-actin to standardize the loading. The primary and secondary antibodies for immunoblotting are shown in Supplementary Information (Tables S1 and S2 ).
3
Protein Separation and Visualization by SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein samples were subjected to SDS-PAGE using Laemmli method (1970). Twenty microliters of the protein samples was mixed with 4 μl loading buffer (0.125 M Tris base, pH 6.8; 20% (w/v) glycerol; 2% (w/v) SDS; 2% (v/v) 2-mercaptoethanol; and 0.01% (w/v) bromophenol blue) and loaded into the wells of the 12% separating gel. Electrophoresis was performed at 100 V over 90 min by using a Mini Protein electrophoresis system (Bio-Rad, USA).
The gels were stained with 0.25% Coomassie Brilliant Blue R-250 (Sigma) in 40% (v/v) methanol and 7% (v/v) acetic acid for 1 h and de-stained with 40% (v/v) methanol and 7% (v/v) acetic acid until the background was clear. Gel scanning and visualization were performed using a densitometer (GS-800, Bio-Rad). The analysis was conducted using UVIDoc Analyzer software.
The gels were stained with 0.25% Coomassie Brilliant Blue R-250 (Sigma) in 40% (v/v) methanol and 7% (v/v) acetic acid for 1 h and de-stained with 40% (v/v) methanol and 7% (v/v) acetic acid until the background was clear. Gel scanning and visualization were performed using a densitometer (GS-800, Bio-Rad). The analysis was conducted using UVIDoc Analyzer software.
4
SDS-PAGE Analysis of Bacterial Biomass
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE was conducted by using a mini-protein electrophoresis system (Bio-Rad Laboratories, Hercules, Canada) according to the Laemmli method. Non-diluted bacterial biomass was mixed in 1:1 ratio with the buffer with and without β-mercaptoethanol. The mixtures were then heated at 95 °C for 5 min and loaded into the gels. The electric current was set at 30 mA. After that, the gels were left in a coloration solution (Coomassie Brilliant Blue R- 250) for 1 h, followed by the initial decoloration for 30 min and the secondary overnight decoloration [19 (link)].
5
Western Blot Analysis of GV Oocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sample of GV oocytes were collected from WT and ClpP cKO female C57BL/6 mice and suspended in lysis buffer [50mM HEPES-KOH (pH 7.5), 100mM KCl, 2mM EDTA, 10% glycerol, 0.1% NP-40, 10mM NaF, 0.25mM Na3VO4, and 50mM ßglycerophosphate] supplemented with complete protease inhibitor (Cat. 04693116001, Roche, Basel, Switzerland). The samples were homogenized and centrifuged at 20,000 g for 20min at 4°C, after which the supernatant was retained for western blotting analysis. The proteins in each sample were separated using 8–16% Bis-Tris gels (Cat. M00659, SurePAGE™, GenScript, Nanjing, China) and a mini protein electrophoresis system (Cat. 1658034, BIO-RAD, CA, USA) following the manufacturer’s instructions. The protein bands were then transferred to polyvinylidene fluoride (PVDF) membranes (Cat. IPVH00010, Immobilon, Millipore, MA, USA) via a Mini Trans-Blot Electrophoretic Transfer Cell (Cat. 1703930, BIO-RAD, CA, USA). The immunoreactive bands were detected and analyzed with a Bio-Rad ChemiDoc MP imaging System (Cat. 12003154, BIO-RAD, CA, USA) in conjunction with the Image Lab Software (Bio-Rad, CA, USA). The relative protein levels in each sample were normalized to ß-Actin to standardize the loading variations. The obtained images were analyzed by image J software for gray value analysis.
6
Quantifying Neuroligin 2 Levels in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein levels were quantified using total brain homogenates from 3 groups of adult male littermates- WT, heterozygous and homozygous. The western blot system used was the standard Bio-Rad mini protein electrophoresis system and the procedure followed the system manual. LiCOR Odyssey Clx was used for protein signal detection. The antibodies used were Rb anti-Neuroligin 2 (1:1000, SYSY 129202), Rb anti-GAPDH (1:10000, Sigma G9545), and Gt anti-Rb 800 (1:15000, P/N 925-32210, P/N 925-32211). Detailed procedures are described in the Additional file 1 .
7
SPI Protein Electrophoresis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Electrophoretic patterns of SPI were visualized by SDS-PAGE, conducted under both reducing (with the addition of β-mercaptoethanol) and non-reducing (without β-mercaptoethanol) conditions. SPI samples were configured at a concentration of 2 mg/mL, and each SPI sample and molecular weight markers were loaded in volumes of 10 μL. A 4% concentrated gel and 12% separated gel were employed for analysis. Gel electrophoresis was performed using a Bio-Rad Mini Protein Electrophoresis System (Bio-Rad Laboratories, Hercules, CA, USA) at 40 V for the concentrated gel and at 80 V for the separated gel. To achieve a clear background, the gel was washed three times with boiling water, stained with rapid staining solution for 30 min, and then decolorized with deionized water for 120 min.
8
SDS-PAGE Analysis of Surimi Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare the protein sample, 9 mL of 5% (w/v) SDS was added to 1 g surimi gels, and the mixture was homogenized for 1 min. The homogenate was incubated in 85 °C water bath for 60 min to dissolve total proteins, followed by centrifuging at 9800 g for 20 min, and the supernatant was retained. Protein concentration of the supernatant was determined by Lowry's method [24] (link) and adjusted to 2 mg·mL−1.
The system used for experiment was Mini-Protein Electrophoresis system (Bio-Rad Laboratories, Inc., USA). Firstly, the protein solution (2 mg·mL−1) was mixed with the loading buffer at 4:1 (protein: buffer), then the mixture was heated at 95 °C for 5 min before loading. Ten microliters of sample were added to each lane. The concentration of separating and stacking gel was 8% and 5%, respectively. At the beginning, voltage was set at 80 V, followed by being elevated to 120 V when the entry of samples was into the stacking gel. Furthermore, the gel was dyed at 37 °C for 90 min in the staining solution (containing 0.125% (w/v) Coomassie brilliant blue R-250), and was destained in the solution containing 25% (v/v) ethyl alcohol and 8% (v/v) acetic acid until the appearance of clear protein bands.
The system used for experiment was Mini-Protein Electrophoresis system (Bio-Rad Laboratories, Inc., USA). Firstly, the protein solution (2 mg·mL−1) was mixed with the loading buffer at 4:1 (protein: buffer), then the mixture was heated at 95 °C for 5 min before loading. Ten microliters of sample were added to each lane. The concentration of separating and stacking gel was 8% and 5%, respectively. At the beginning, voltage was set at 80 V, followed by being elevated to 120 V when the entry of samples was into the stacking gel. Furthermore, the gel was dyed at 37 °C for 90 min in the staining solution (containing 0.125% (w/v) Coomassie brilliant blue R-250), and was destained in the solution containing 25% (v/v) ethyl alcohol and 8% (v/v) acetic acid until the appearance of clear protein bands.
9
Protein Analysis of Surimi Gels by SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein content of surimi gels eluted in 10 % (w/w) SDS was determined by SDS-PAGE using precast polyacrylamide gels (5 % to 20 %, Sobaco) according to the method reported by Laemmli et al. [28] (link) and Petcharat et al. [5] . The supernatant extracted from each treatment group was adjusted to 2 mg/mL, then an 8 μL sample was loaded onto a polypropylene gel. In brief, about 3.0 g gel sample was evenly distributed in 27 mL 10 % (w/w) SDS solution, and then heated at 85 °C for 30 min. The mixture was centrifuged at 10, 000 × g for 20 min, and the supernatant was retained. The protein composition was performed on a precast polyacrylamide gel (5 % ∼ 20 %, Sobaco), and the protein sample was loaded into 10 μL. Separating gels were run at 80 V and stacking gels were run at 120 V using a Mini-Protein Electrophoresis System (Bio-Rad Laboratories Inc., Hercules, CA, USA). Imaging was performed after overnight destaining with Coomassie brilliant blue R-250.
10
Comprehensive Research Equipment Setup
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Model 550 microplate reader, miniprotein electrophoresis system, and miniprotein transfer membrane system were purchased from BIO-RAD (California, USA). KD paraffin microtome was purchased from Shanghai fourth medical instrument factory (Shanghai, China). OLYMPUS bx-41 microscope was purchased from OLYMPUS (Tokyo, Japan). AlC-CWB numerical control constant temperature circulating water tank was purchased from Shanghai Alcott Biotech Co., Ltd. (Shanghai, China). Multichannel microsampling system was purchased from Inbio Life Science Instrument Co., Ltd. (Wuhan, China). Glass electrode drawing instrument was purchased from MDI (USA). Leica TCS Sp8 confocal laser scanning microscope was purchased from Leica (Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!