To assess purity, a polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate (SDS-PAGE) under reducing conditions was performed, in a Mini-PROTEAN Tetra Cell (Biorad), mixing samples with sample buffer and loading 20 µL of samples and 5 µL of the Broad Multi Color Pre-Stained Protein Standard (Genscript) into the wells of ExpressPlus PAGE Gels (Genscript) and using Tris-MOPS-SDS buffer (Genscript) as running buffer, in a two-step running starting at 60V for 30 min and then changed to 110 V for 1 hour and 30 minutes, with a PowerPac Basic power supply (Biorad). Gels were stained with 0.25% Coomassie Blue R-250 for 4 hours and distained with a solution containing 10% acetic acid, 30% methanol, and 60% water, applying four washes of 30 min each. Both staining and distaining were performed using a rocking platform settled at 14 oscillations/min.
Broad multi color pre stained protein standard
Manufactured by GenScript
Sourced in United Kingdom
The Broad Multi Color Pre-Stained Protein Standard is a laboratory tool used for molecular weight determination of proteins in SDS-PAGE analysis. The product contains a mixture of pre-stained proteins with a broad range of molecular weights, providing a visual size reference during electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tetra cell, by Bio-Rad (3 mentions)
Surepage precast gel, by GenScript (2 mentions)
Mes sds running buffer, by GenScript (2 mentions)
Instantblue ultrafast protein stain, by Abcam (2 mentions)
Ppsq 53a, by Shimadzu (2 mentions)
Expressplus page gel, by GenScript (1 mentions)
Powerpac basic power supply, by Bio-Rad (1 mentions)
Surepage, by GenScript (1 mentions)
Instantblue coomassie protein stain, by Abcam (1 mentions)
Pierce bca protein assay kit 23225, by Thermo Fisher Scientific (1 mentions)
Tris mops sds running buffer, by GenScript (1 mentions)
4 protocols using broad multi color pre stained protein standard
1
SDS-PAGE Analysis of Protein Purity
2
Proteomic Profiling of Spermatozoa
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fresh semen was washed three times using phosphate buffered saline (PBS) and centrifuged at 1,800 rpm. Following the manual’s instructions, the spermatozoa pellet was extracted using PRO-PREPTM protein extraction solution (iNtRON Biotechnologi, Korea). The total soluble protein concentration of the sample was measured before analysis by SDS-PAGE. The colorimetric detection and quantification of total protein were conducted using the bicinchoninic acid (BCA) method, employing the Pierce™ BCA Protein Assay Kit 23225 from Thermo Scientific™, USA. SDS-PAGE analysis was performed to determine the protein profile based on MW, which is represented as bands on the gels. Protein separation was carried out using SDS-PAGE using SurePAGE™, Bis-Tris, 10 × 8 cm, 12 wells, 4%–20% gradient gel (M00656; GenScript) (SurePAGE, Genscript Biotech Corp. Hongkong) with Broad Multi Color Pre-Stained Protein Standard (M00624; GenScript) with a MW range of ~5–270 kDa and Tris-MOPS-SDS Running Buffer (M00138; GenScript) with a voltage of 140 V and a current of 75 mA for 55 min. The gel was then stained using InstantBlue® Coomassie Protein Stain (ab119211; abcam). The differential intensity of individual protein bands was assessed by conducting ratio analysis with the aid of ImageJ software [9] .
3
Activation of Globupain Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For activation of globupain and substitution variants (Table 1 ), a purified enzyme sample (< 5 mg/mL) was incubated at 75°C for up to 4.5 h in 20 mM tri-sodium citrate dihydrate, 150 mM NaCl, pH 5.5 (at room temperature) with 2.5 mM DTT and 1 mM CaCl2, respectively (activation buffer). To investigate if globupain could in-trans activate, 10 μg of activated WT globupain was mixed with 10 μg of inactive C185A variant. The number and size of cleavage products were assessed by visualization of protein bands on 8–16% SurePAGE precast gels (GenScript) using MES SDS running buffer (GenScript) in a Bio-Rad Mini-PROTEAN Tetra Cell (BioRad, Hercules, CA, USA). For sample preparation, 4´ lithium dodecyl sulfate (LDS) sample buffer (GenScript) with 2-mercaptoethanol was mixed with the protein sample, followed by denaturing at 95°C for 10 min. Gels were stained with InstantBlue™ ultrafast protein stain (Abcam, Cambridge, United Kingdom), and the size of bands was indicated by broad multi-color pre-stained protein standard (GenScript). Edman sequencing was performed on a Shimadzu PPSQ-53A at the Iowa State University Protein Facility, USA.
4
Activation and Cleavage Analysis of Globupain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For activation of globupain and substitution variants (Table 1 ), purified enzyme (< 5 mg/mL) was incubated at 75°C for up to 4.5 h in 20 mM tri-sodium citrate dihydrate, 150 mM NaCl, pH 5.5 (at RT) with 2.5 mM DTT and 1 mM CaCl2, respectively (activation buffer). To investigate if globupain could in-trans activate, 10 μg of activated WT globupain was mixed with 10 μg of inactive C185A variant. The number and size of cleavage products were assessed by visualization of protein bands on 8%–16% SurePAGE precast gels (GenScript) using MES SDS running buffer (GenScript) in a Bio-Rad Mini-PROTEAN Tetra Cell (BioRad, Hercules, CA, United States). For sample preparation, 4′ lithium dodecyl sulfate (LDS) sample buffer (GenScript) with 2-mercaptoethanol was mixed with the protein sample, followed by denaturing at 95°C for 10 min. Gels were stained with InstantBlue™ ultrafast protein stain (Abcam, Cambridge, United Kingdom), and the size of bands was indicated by broad multi-color pre-stained protein standard (GenScript). Edman sequencing was performed on a Shimadzu PPSQ-53A at the Iowa State University Protein Facility, United States.
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!