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Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at <t>4°C</t> with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in <t>TBST.</t> The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.
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1) Product Images from "Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products"

Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004241

Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.
Figure Legend Snippet: Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.

Techniques Used: Western Blot, SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, High Performance Liquid Chromatography

Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.
Figure Legend Snippet: Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.

Techniques Used: SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, Protein Concentration

Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.
Figure Legend Snippet: Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.

Techniques Used: Western Blot, SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, High Performance Liquid Chromatography

Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.
Figure Legend Snippet: Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.

Techniques Used: SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, Protein Concentration

2) Product Images from "Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products"

Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004241

Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.
Figure Legend Snippet: Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.

Techniques Used: Western Blot, SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, High Performance Liquid Chromatography

Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.
Figure Legend Snippet: Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.

Techniques Used: SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, Protein Concentration

Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.
Figure Legend Snippet: Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.

Techniques Used: Western Blot, SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, High Performance Liquid Chromatography

Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.
Figure Legend Snippet: Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.

Techniques Used: SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, Protein Concentration

3) Product Images from "Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products"

Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004241

Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.
Figure Legend Snippet: Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.

Techniques Used: Western Blot, SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, High Performance Liquid Chromatography

Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.
Figure Legend Snippet: Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.

Techniques Used: SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, Protein Concentration

4) Product Images from "Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products"

Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004241

Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.
Figure Legend Snippet: Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.

Techniques Used: Western Blot, SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, High Performance Liquid Chromatography

Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.
Figure Legend Snippet: Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.

Techniques Used: SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, Protein Concentration

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Article Snippet: .. The membranes were washed once for 5 min, 10 min and 15 min each in TBST, incubated for 1h in secondary antibody (Jackson Immuno-research, sheep anti-mouse-HRP, 1∶5000) at room temperature for MSH2, MSH3, MSH6 and for actin, and washed one time for 5 min, 10 min and 15 min each in TBST. .. Antibody binding was visualized using ECL™ Western blotting analysis system and ECL plus Western blotting detection system (Amersham).

Article Title: Vancomycin does not affect the enzymatic activities of purified VanSA
Article Snippet: .. The membrane was washed 3x10 mins with TBST, and then incubated for 1 hour at room temperature with a 1:1000 dilution of goat anti-rabbit secondary antibody (Jackson ImmunoResearch HRP-GARIgG 111-035-003) in 1% milk. .. Finally, the membrane was washed 3x10 mins with TBST before reaction with peroxidase substrate solution for enhanced chemiluminescence (Pierce 32209) and visualized with film.

Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products
Article Snippet: .. Membranes were blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST, and then incubated at room temperature for 2 hr with affinity purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. .. The membranes were washed again with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr.

Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products
Article Snippet: .. The membranes were washed again with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. .. The membranes were washed and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed.

Article Title: Novel Mutations in TARDBP (TDP-43) in Patients with Familial Amyotrophic Lateral Sclerosis
Article Snippet: .. Membranes were washed three times each for 10 minutes with TBST and then incubated with anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (1∶2000; Jackson ImmunoResearch, West Grove, PA) for 1 hour. .. Membranes were then washed three times each for 10 minutes, and protein expression was visualized by ECL treatment and exposure to film.

Fluorescence In Situ Hybridization:

Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products
Article Snippet: .. Membranes were blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST, and then incubated at room temperature for 2 hr with affinity purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. .. The membranes were washed again with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr.

Affinity Purification:

Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products
Article Snippet: .. Membranes were blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST, and then incubated at room temperature for 2 hr with affinity purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. .. The membranes were washed again with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr.

Protein Concentration:

Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products
Article Snippet: .. Membranes were blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST, and then incubated at room temperature for 2 hr with affinity purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. .. The membranes were washed again with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr.

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    Jackson Immuno tbst
    Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at <t>4°C</t> with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in <t>TBST.</t> The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.
    Tbst, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 92/100, based on 1045 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.

    Journal: PLoS ONE

    Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

    doi: 10.1371/journal.pone.0004241

    Figure Lengend Snippet: Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis. Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.

    Article Snippet: Membranes were blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST, and then incubated at room temperature for 2 hr with affinity purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration.

    Techniques: Western Blot, SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, High Performance Liquid Chromatography

    Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.

    Journal: PLoS ONE

    Article Title: Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

    doi: 10.1371/journal.pone.0004241

    Figure Lengend Snippet: Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies. Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.

    Article Snippet: Membranes were blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST, and then incubated at room temperature for 2 hr with affinity purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration.

    Techniques: SDS Page, Fluorescence In Situ Hybridization, Incubation, Affinity Purification, Protein Concentration