To prepare the membrane-associated human GLUT8, the pcDNA3.1 vector (Invitrogen) encoding a cDNA fragment of human GLUT8 was transiently transfected into FreeStyle293 cells. After 3 days of incubation, cells were washed with phosphate-buffered saline (PBS) and suspended in buffer A, which contained 50 mM HEPES (pH 7.4), 1 mM EDTA. The cells were homogenized and centrifuged at 950×g for 10 min at 4 °C, after which the supernatant was recovered. Total membrane fractions were isolated by ultracentrifugation at 140,000×g for 60 min at 4 °C, and resuspended in buffer A. Membrane fractions were frozen at −80 °C until use.
Freestyle293 cells
FreeStyle293 cells are a human embryonic kidney (HEK) cell line designed for high-yield protein production in suspension culture. These cells are adapted for growth in serum-free media and are suitable for transient transfection, facilitating the rapid generation of recombinant proteins.
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14 protocols using freestyle293 cells
Preparation of Human Protein Library
Engineered Mouse PlexinA1/A2 Sema Domain Protein
Example 22
A mouse PlexinA1/A2 sema domain chimeric protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and a sequence from arginine at position 459 to serine at position 510 of the mouse PlexinA2 sema domain protein, the FLAG tag (SEQ ID NO: 5), and the termination codon were added to a sequence after isoleucine at position 458 of the mouse PlexinA1 sema domain protein. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 40. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1/A2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.
Recombinant CCHFV GP38 Protein Production
Stabilized HIV-1 gp140 Env Trimer Production
Recombinant CCHFV GP38 Protein Production
Soluble mouse PlexinA1 protein production
Example 4
Soluble mouse PlexinA1 protein was designed on the basis of the amino acid sequence of NCBI Reference Sequence NP_032907.1 (SEQ ID NO: 52) up to the extracellular domain. The signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag sequence (SEQ ID NO: 5) was inserted at the C-terminus. The prepared amino acid sequence is shown in (SEQ ID NO: 16). The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the soluble mouse PlexinA1 protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.
Production of Mouse PlexinA1 Sema Domain
Example 20
A mouse PlexinA1 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP 032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 512. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 38. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.
Designing Chimeric Plexin Sema Domains
Example 22
A mouse PlexinA1/A2 sema domain chimeric protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and a sequence from arginine at position 459 to serine at position 510 of the mouse PlexinA2 sema domain protein, the FLAG tag (SEQ ID NO: 5), and the termination codon were added to a sequence after isoleucine at position 458 of the mouse PlexinA1 sema domain protein. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 40. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1/A2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.
Recombinant Mouse PlexinA1 Sema Domain
Example 20
A mouse PlexinA1 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 512. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 38. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.
Purification of Mouse PlexinA2 Sema Domain
Example 21
A mouse PlexinA2 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP 032908.2 (SEQ ID NO: 53) to encode a sequence in which the signal peptide (from the N-terminus to glycine at position 31) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 510. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 39. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.
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