Bestrophin 1 Antibody (E6-6) - BSA Free

A: This antibody was validated in IHC in the reference PMID 11050159. Generally we recommend using antigen retrieval with citrate buffer and incubating for 1 hour at RT. Some antigens may be tricky and require overnight incubation. I imagine the specific protocol can be found in the reference, however.

A: Here is both the recipe we use for RIPA and a good protocol for tissue lysate prep. RIPA buffer: 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 RIPA buffer is also useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations/pull down assays. Preparation of lysate from tissues Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases. Place the tissue in round-bottom microfuge tubes or Eppendorf tubes and immerse in liquid nitrogen to snap freeze. Store samples at -80C for later use or keep on ice for immediate homogenization. For a approximately 5 mg piece of tissue, add approximately 300 ul lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2x300 ul lysis buffer, then maintain constant agitation for 2 hours at 4C (e.g place on an orbital shaker in the fridge). Volumes of lysis buffer must be determined in relation to the amount of tissue present (protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. The minimum concentration is 0.1 mg/ml, optimal concentration is 1-5 mg/ml). Centrifuge for 20 min at 12000 rpm at 4C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice; discard the pellet.

A: For our product NB300-164, this antibody is provided as unpurified ascites. Therefore, the antibody concentration has not been determined.

A: The total protein concentration of the ascites (Lot# B-3) is 11.7 mg/ml.

A: This antibody was validated in IHC in the reference PMID 11050159. Generally we recommend using antigen retrieval with citrate buffer and incubating for 1 hour at RT. Some antigens may be tricky and require overnight incubation. I imagine the specific protocol can be found in the reference, however.

A: Here is both the recipe we use for RIPA and a good protocol for tissue lysate prep. RIPA buffer: 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 RIPA buffer is also useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations/pull down assays. Preparation of lysate from tissues Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases. Place the tissue in round-bottom microfuge tubes or Eppendorf tubes and immerse in liquid nitrogen to snap freeze. Store samples at -80C for later use or keep on ice for immediate homogenization. For a approximately 5 mg piece of tissue, add approximately 300 ul lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2x300 ul lysis buffer, then maintain constant agitation for 2 hours at 4C (e.g place on an orbital shaker in the fridge). Volumes of lysis buffer must be determined in relation to the amount of tissue present (protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. The minimum concentration is 0.1 mg/ml, optimal concentration is 1-5 mg/ml). Centrifuge for 20 min at 12000 rpm at 4C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice; discard the pellet.

A: For our product NB300-164, this antibody is provided as unpurified ascites. Therefore, the antibody concentration has not been determined.

A: The total protein concentration of the ascites (Lot# B-3) is 11.7 mg/ml.

A: This antibody was validated in IHC in the reference PMID 11050159. Generally we recommend using antigen retrieval with citrate buffer and incubating for 1 hour at RT. Some antigens may be tricky and require overnight incubation. I imagine the specific protocol can be found in the reference, however.

A: Here is both the recipe we use for RIPA and a good protocol for tissue lysate prep. RIPA buffer: 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 RIPA buffer is also useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations/pull down assays. Preparation of lysate from tissues Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases. Place the tissue in round-bottom microfuge tubes or Eppendorf tubes and immerse in liquid nitrogen to snap freeze. Store samples at -80C for later use or keep on ice for immediate homogenization. For a approximately 5 mg piece of tissue, add approximately 300 ul lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2x300 ul lysis buffer, then maintain constant agitation for 2 hours at 4C (e.g place on an orbital shaker in the fridge). Volumes of lysis buffer must be determined in relation to the amount of tissue present (protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. The minimum concentration is 0.1 mg/ml, optimal concentration is 1-5 mg/ml). Centrifuge for 20 min at 12000 rpm at 4C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice; discard the pellet.

A: For our product NB300-164, this antibody is provided as unpurified ascites. Therefore, the antibody concentration has not been determined.

A: The total protein concentration of the ascites (Lot# B-3) is 11.7 mg/ml.

A: This antibody was validated in IHC in the reference PMID 11050159. Generally we recommend using antigen retrieval with citrate buffer and incubating for 1 hour at RT. Some antigens may be tricky and require overnight incubation. I imagine the specific protocol can be found in the reference, however.

A: Here is both the recipe we use for RIPA and a good protocol for tissue lysate prep. RIPA buffer: 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 RIPA buffer is also useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations/pull down assays. Preparation of lysate from tissues Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases. Place the tissue in round-bottom microfuge tubes or Eppendorf tubes and immerse in liquid nitrogen to snap freeze. Store samples at -80C for later use or keep on ice for immediate homogenization. For a approximately 5 mg piece of tissue, add approximately 300 ul lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2x300 ul lysis buffer, then maintain constant agitation for 2 hours at 4C (e.g place on an orbital shaker in the fridge). Volumes of lysis buffer must be determined in relation to the amount of tissue present (protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. The minimum concentration is 0.1 mg/ml, optimal concentration is 1-5 mg/ml). Centrifuge for 20 min at 12000 rpm at 4C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice; discard the pellet.

A: For our product NB300-164, this antibody is provided as unpurified ascites. Therefore, the antibody concentration has not been determined.

A: The total protein concentration of the ascites (Lot# B-3) is 11.7 mg/ml.