GST-tagged proteins were expressed in Escherichia coli BL21. The BL21 cells were grown to an OD600 of 0.6; expressed protein was induced with isopropyl-β-d-thiogalactoside (IPTG) to a final concentration of 1 mM at 25°C for 4–6 hr. The bacterial pellets were resuspended in lysis buffer (1x TBS supplemented with 1% Triton, 1 mM EDTA, 1 mM PMSF, and cOmplete protease inhibitor cocktail tablets). After sonication and centrifugation, the crude extract was purified using Glutathione-Sepharose 4 Fast Flow beads (GE Healthcare). Mouse brains were homogenized in Binding Buffer (25 mM HEPES, 10 mM NaCl, 0.01% Triton, 5% Glycerol, 1 mM DTT, pH7.5, and cOmplete protease inhibitor cocktail tablets). The homogenates were centrifuged at 15,000 × g for 20 min. HEK293T cells were lysed in Binding Buffer and centrifuged at 15,000 × g for 20 min. The supernatant was carefully collected. GST beads coated with GST, GST-SNPH, or its truncated mutants were mixed with the brain homogenate or HEK293T cell supernatants and incubated for 3 hr with gentle agitation. The beads were then extensively washed with 1x TBS supplemented with 0.1% Triton. Next, the beads were dissolved in LDS-PAGE sample buffer (Thermo Fisher Scientific) and heated at 75°C for 10 min. Proteins were resolved by Bis-Tris NuPAGE and processed for immunoblot analysis or Coomassie Blue Staining.
Lds page sample buffer
Manufactured by Thermo Fisher Scientific
LDS-PAGE sample buffer is a laboratory reagent used to prepare protein samples for electrophoresis analysis. It is designed to denature and solubilize proteins, allowing for their separation and detection in a polyacrylamide gel.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using lds page sample buffer
1
Purification and Characterization of GST-Tagged Proteins
2
Purification and Characterization of GST-Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GST-tagged proteins were expressed in Escherichia coli BL21. The BL21 cells were grown to an OD600 of 0.6; expressed protein was induced with isopropyl-β-d-thiogalactoside (IPTG) to a final concentration of 1 mM at 25°C for 4–6 hr. The bacterial pellets were resuspended in lysis buffer (1x TBS supplemented with 1% Triton, 1 mM EDTA, 1 mM PMSF, and cOmplete protease inhibitor cocktail tablets). After sonication and centrifugation, the crude extract was purified using Glutathione-Sepharose 4 Fast Flow beads (GE Healthcare). Mouse brains were homogenized in Binding Buffer (25 mM HEPES, 10 mM NaCl, 0.01% Triton, 5% Glycerol, 1 mM DTT, pH7.5, and cOmplete protease inhibitor cocktail tablets). The homogenates were centrifuged at 15,000 × g for 20 min. HEK293T cells were lysed in Binding Buffer and centrifuged at 15,000 × g for 20 min. The supernatant was carefully collected. GST beads coated with GST, GST-SNPH, or its truncated mutants were mixed with the brain homogenate or HEK293T cell supernatants and incubated for 3 hr with gentle agitation. The beads were then extensively washed with 1x TBS supplemented with 0.1% Triton. Next, the beads were dissolved in LDS-PAGE sample buffer (Thermo Fisher Scientific) and heated at 75°C for 10 min. Proteins were resolved by Bis-Tris NuPAGE and processed for immunoblot analysis or Coomassie Blue Staining.
3
Protein Extraction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with PBS and lysed with ice-cold lithium dodecyl sulfate (LDS)-PAGE sample buffer (Invitrogen) containing 250 U/μl benzonase (Accelagen), 1 μg/ml pepstatin A, 5 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, and 5.4 mM sodium orthovanadate for 10 min on ice. β-Mercaptoethanol was then added to a final concentration of 0.36 M at 95°C for 5 min, followed by centrifugation at 20,000 × g at 4°C for 10 min. Protein concentrations were determined using the Pierce 660-nm protein assay reagent containing ionic detergent compatibility reagent and read at 660 nm in a SpectraMax M2 plate reader.
4
Cell Lysis and Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with Hank’s balanced salt solution (HBSS) and lysed with ice-cold lithium dodecyl sulfate (LDS)-PAGE sample buffer (Invitrogen) containing 250 U/μL benzonase (Accelagen), 1 μg/mL pepstatin A, 5 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, and 5.4 mM sodium orthovanadate for 10 min on ice. β-Mercaptoethanol was then added to a final concentration of 0.36 M. Lysates were incubated at 95°C for 5 min and then clarified by centrifugation at 20,000 × g at 4°C for 10 min. Protein concentrations were determined using the Pierce 660-nm protein assay reagent containing ionic detergent compatibility reagent and read at 660 nm in a SpectraMax M2 plate reader (Molecular Devices).
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!