To the isolated amyloid plaques, 50 μl of 70% FA, with 5 mM EDTA, was added. The samples were sonicated for 5 min and incubated for 1 hour at 24°C. The samples were then neutralized to pH 7 using 0.5 M tris buffer. Aβ peptides were than purified through immunoprecipitation using Aβ-specific antibodies (antibodies 6E10 and 4G8, Signet Laboratories), coupled to magnetic Dynabeads M-280 Sheep Anti-Mouse (Invitrogen), as described previously (59 (link)). The supernatant was collected and dried through lyophilization.
M 280 sheep anti mouse dynabeads
Manufactured by Thermo Fisher Scientific
Sourced in United States
The M-280 sheep anti-mouse Dynabeads are uniform, superparamagnetic polymer beads coated with sheep polyclonal antibodies specific for mouse immunoglobulins. They are designed for the rapid isolation and purification of mouse immunoglobulins and mouse antibody-producing cells from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (3 mentions)
Anti v5 antibody, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Protein a magnetic beads, by Thermo Fisher Scientific (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Palmitate, by Merck Group (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Mem a, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin l glutamine, by Thermo Fisher Scientific (1 mentions)
5 protocols using m 280 sheep anti mouse dynabeads
1
Amyloid Plaque Extraction and Purification
2
Isoproterenol and Palmitate Stimulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isoproterenol (cat # I6504), IBMX (cat # I5879), Palmitate (cat # P0500-100G), Rp-Adenosine 3′,5′-cylic monophophorothioate triethylammonium salt (cat # A165-1MG), cycloheximide (cat # C1988) and oleic acid (cat # O1008) were from Sigma (St. Louis, MO, USA). PKA inhibitors PKI, 14-22 Amide, cell permeable myristoylated was from Calbiochem (cat # 476485).
Oleate complexed with albumin 5.5:1 molar ratio for cell culture treatments was prepared as previously described50 (link) as follows: oleic acid was solubilized with 1 M sodium hydroxide at 60 °C, then the sodium-oleate solution was bound to fatty acid-free bovine serum albumin (Fitzgerald cat # 30-AB79) at a molar ratio of 5.5:1.
DMEM, MEM-a, RPMI1640, fetal bovine serum (FBS) and penicillin/streptomycin/L -glutamine, Lipofectamine 2000 were from Invitrogen Corp. (Carlsbad, CA, USA).
Dynabeads M280 Sheep anti-Mouse (cat # 11201D) and Sheep anti-Rabbit (cat # 2016D6) were from Invitrogen. Protein A magnetic beads were from Thermo Scientific (cat # 21348).
Oleate complexed with albumin 5.5:1 molar ratio for cell culture treatments was prepared as previously described50 (link) as follows: oleic acid was solubilized with 1 M sodium hydroxide at 60 °C, then the sodium-oleate solution was bound to fatty acid-free bovine serum albumin (Fitzgerald cat # 30-AB79) at a molar ratio of 5.5:1.
DMEM, MEM-a, RPMI1640, fetal bovine serum (FBS) and penicillin/streptomycin/
Dynabeads M280 Sheep anti-Mouse (cat # 11201D) and Sheep anti-Rabbit (cat # 2016D6) were from Invitrogen. Protein A magnetic beads were from Thermo Scientific (cat # 21348).
3
Mapping MondoA-V5 Binding Sites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MondoA-V5 was transfected into HeLa cells. Chromatin was cross-linked and sheared as described [19 (link)]. Chromatin was incubated overnight with anti-V5 antibody (Thermo Fisher) or mouse IgG (Sigma Aldrich). M-280 sheep anti-mouse Dynabeads (Thermo Fisher) were used to capture and purify immunocomplexes. DNA was purified using a QIAquick PCR Purification Kit (Qiagen) and analyzed using quantitative PCR as described above. Primers were previously described [20 (link)].
4
Chromatin Immunoprecipitation and Promoter Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MondoA-V5 was transfected into HeLa cells. Chromatin was cross-linked and sheared as described (19) . Chromatin was incubated overnight with anti-V5 antibody (Thermo Fisher) or mouse IgG (Sigma Aldrich). M-280 sheep anti-mouse Dynabeads (Thermo Fisher) were used to capture and purify immunocomplexes. DNA was purified using a QIAquick PCR Purification Kit (Qiagen) and analyzed using quantitative PCR as described above. Primers were previously described (20) .
Promoter activity assay TXNIP promoter luciferase assays were performed as described previously (21) .
Briefly, cells were transfected with a TXNIP promoter luciferase construct and a CMVdriven b-galactosidase construct. Following treatments, luciferase and b-galactosidase activities were determined according to manufacturer's recommendations (Promega, Tropix). Luciferase activity was normalized to b-galactosidase to control for differences in transfection efficiency.
Promoter activity assay TXNIP promoter luciferase assays were performed as described previously (21) .
Briefly, cells were transfected with a TXNIP promoter luciferase construct and a CMVdriven b-galactosidase construct. Following treatments, luciferase and b-galactosidase activities were determined according to manufacturer's recommendations (Promega, Tropix). Luciferase activity was normalized to b-galactosidase to control for differences in transfection efficiency.
5
Chromatin Immunoprecipitation and Promoter Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MondoA-V5 was transfected into HeLa cells. Chromatin was cross-linked and sheared as described (19) . Chromatin was incubated overnight with anti-V5 antibody (Thermo Fisher) or mouse IgG (Sigma Aldrich). M-280 sheep anti-mouse Dynabeads (Thermo Fisher) were used to capture and purify immunocomplexes. DNA was purified using a QIAquick PCR Purification Kit (Qiagen) and analyzed using quantitative PCR as described above. Primers were previously described (20) .
Promoter activity assay TXNIP promoter luciferase assays were performed as described previously (21) .
Briefly, cells were transfected with a TXNIP promoter luciferase construct and a CMVdriven b-galactosidase construct. Following treatments, luciferase and b-galactosidase activities were determined according to manufacturer's recommendations (Promega, Tropix). Luciferase activity was normalized to b-galactosidase to control for differences in transfection efficiency.
Promoter activity assay TXNIP promoter luciferase assays were performed as described previously (21) .
Briefly, cells were transfected with a TXNIP promoter luciferase construct and a CMVdriven b-galactosidase construct. Following treatments, luciferase and b-galactosidase activities were determined according to manufacturer's recommendations (Promega, Tropix). Luciferase activity was normalized to b-galactosidase to control for differences in transfection efficiency.
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!