Polyclonal Antibody Generation and Validation for BAI1

An anti-BAI1 rabbit polyclonal was generated (Covance, Princeton, NJ) against amino acids 1180–1584 of human BAI1 and affinity purified using the antigen purified as a thrombin-cleaved GST-fusion coupled to UltraLink BioSupport (Thermo Scientific, Waltham, MA). We described this antibody previously (Duman et al., 2013 (link)); BAI1 is only detected by this antibody in Western blots of Cos-7 cells when they are exogenously expressing BAI1. The immunoreactivity of this antibody for neurons is strongly decreased by BAI1 Kd. This antibody was used at 0.001 mg/ml for IHC, ICC, and Western blotting and was reused at least 5X. For immunoprecipitation, 1 µg per 1.3 × 10⁶ neurons in culture was used. Rabbit anti-BAI11 N-terminal polyclonal (H-270, sc-66815), rabbit anti-Bcr polyclonal (N-20, sc-885), mouse anti-Abr monoclonal IgG₁ (24, sc-135821), and mouse monoclonal anti-c-myc IgG₁ (9E10, sc-40) were obtained from Santa Cruz Biotechnology (Dallas, TX) and used at 1:1000 dilution for Westerns and ICC. DMSO (D2650) was obtained from Sigma (St. Louis, MO). Tetradotoxin, narciclasine, D-AP5, and Y-27632 dihydrochloride were obtained from Tocris (Minneapolis, MN). Potassium chloride (P217) was obtained from Fisher Scientific (Pittsburgh, PA).

Free full text: Click here

Duman J.G., Mulherkar S., Tu Y.K., Erikson K.C., Tzeng C.P., Mavratsas V.C., Ho T.S, & Tolias K.F. (2019). The adhesion-GPCR BAI1 shapes dendritic arbors via Bcr-mediated RhoA activation causing late growth arrest. eLife, 8, e47566.

Publication 2019

UltraLink BioSupport DMSO (D2650) narciclasine D-AP5 Y-27632 dihydrochloride

Amino acids Antibody Antigen Cos 7 cells Dilution Dmso Human Igg1 Immunoprecipitation Mouse Narciclasine Neurons Per 1 Potassium chloride Rabbit Tetradotoxin Thrombin Western blots Y 27632

Corresponding Organization :

Other organizations : Baylor College of Medicine, Rice University

Top 5 similar protocols

Variable analysis

independent variables

Exogenous expression of BAI1 in Cos-7 cells
BAI1 knockdown (Kd) in neurons

dependent variables

BAI1 detection by the anti-BAI1 antibody in Western blots
Immunoreactivity of the anti-BAI1 antibody for neurons

control variables

Concentration of anti-BAI1 antibody used for IHC, ICC, and Western blotting (0.001 mg/ml)
Concentration of anti-BAI1 antibody used for immunoprecipitation (1 µg per 1.3 × 10^6 neurons)
Dilution of anti-BAI11 N-terminal polyclonal, anti-Bcr polyclonal, anti-Abr monoclonal, and anti-c-myc monoclonal antibodies (1:1000)

positive controls

BAI1 detection by the anti-BAI1 antibody in Western blots of Cos-7 cells exogenously expressing BAI1

negative controls

Decreased immunoreactivity of the anti-BAI1 antibody for neurons due to BAI1 knockdown

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

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?

Revolutionizing how scientists
search and build protocols!