Journal: PLoS ONE

Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

doi: 10.1371/journal.pone.0038313

Figure Lengend Snippet: (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid, probed with anti-PSD-95 (IgG2a, blue), anti-Kv1.2 (IgG2b, red) and anti-Kv2.1 (IgG1, green), and SCS 2°Abs. Multicolor panel is original immunoblot; single color panels are images of separated colors. Lane to left shows molecular weight standards in kDa. Note differential post-translational modification of target proteins in brain versus heterologous cells alters their relative electrophoretic mobility. B–E. Images show specific and non-overlapping labeling for (B) Kv4.2 (green), (C) QKI (red), (D) and BK channels (blue), and (E) merge of all three, in a rat brain section, showing the region containing the entire cerebellum. Inset in E shows boxed area of cerebellar cortex. Labels mark the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). Scale bar on Panel E = 500 µm.

Article Snippet: The generation and validation of anti-PSD-95 mAb K28/43 (IgG2a) , anti-KChIP1 mAb K55/7 (IgG1) and anti-Kv4.2 mAb K57/1 (IgG1) , anti-Caspr/Paranodin mAb K65/35 (IgG1) , anti-Kv2.1 mAb K89/34 (IgG1) , anti-BK channel mAb L6/60 (IgG2a) were described previously. mAbs against QKI (N147/6, IgG2b), Ankyrin-G (N106/36, IgG2a) and GFAP (N206A/8, IgG1) were obtained from the UC Davis/NIH NeuroMab Facility, which also distributes K14/16, K28/43, K55/7, K57/1, K65/35, K89/34 and L6/60.

Techniques: Western Blot, Transfection, Expressing, Control, Plasmid Preparation, Molecular Weight, Modification, Labeling

Journal: PLoS ONE

Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

doi: 10.1371/journal.pone.0038313

Figure Lengend Snippet: Rat brain sections were labeled with the same concentrations of a single mAb, and a rabbit anti-Kv2.1 pAb, followed by detection with SCS (left column) or HL (right column) 2°Abs, (red), and anti-rabbit IgG (green), each at 1 µg/ml. Top row: anti-Kv4.2 IgG1; middle row: anti-BK channel IgG2a; and bottom row: anti-Kv1.2 IgG2b. Each row was imaged at the same exposure times. Scale bar = 50 µm for panels in top two rows, and 25 µm for panels in bottom row.

Article Snippet: The generation and validation of anti-PSD-95 mAb K28/43 (IgG2a) , anti-KChIP1 mAb K55/7 (IgG1) and anti-Kv4.2 mAb K57/1 (IgG1) , anti-Caspr/Paranodin mAb K65/35 (IgG1) , anti-Kv2.1 mAb K89/34 (IgG1) , anti-BK channel mAb L6/60 (IgG2a) were described previously. mAbs against QKI (N147/6, IgG2b), Ankyrin-G (N106/36, IgG2a) and GFAP (N206A/8, IgG1) were obtained from the UC Davis/NIH NeuroMab Facility, which also distributes K14/16, K28/43, K55/7, K57/1, K65/35, K89/34 and L6/60.

Techniques: Labeling

Journal: PLoS ONE

Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

doi: 10.1371/journal.pone.0038313

Figure Lengend Snippet: (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a>IgG2b>IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in . Changes in red∶green tint reflect bias of HL for (more red) IgG2a>IgG2b>IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.

Article Snippet: The generation and validation of anti-PSD-95 mAb K28/43 (IgG2a) , anti-KChIP1 mAb K55/7 (IgG1) and anti-Kv4.2 mAb K57/1 (IgG1) , anti-Caspr/Paranodin mAb K65/35 (IgG1) , anti-Kv2.1 mAb K89/34 (IgG1) , anti-BK channel mAb L6/60 (IgG2a) were described previously. mAbs against QKI (N147/6, IgG2b), Ankyrin-G (N106/36, IgG2a) and GFAP (N206A/8, IgG1) were obtained from the UC Davis/NIH NeuroMab Facility, which also distributes K14/16, K28/43, K55/7, K57/1, K65/35, K89/34 and L6/60.

Techniques: Western Blot, Transfection, Expressing, Control, Plasmid Preparation, Labeling, Molecular Weight, Immunofluorescence, Quantitation Assay, Immunocytochemistry

Journal: PLoS ONE

Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

doi: 10.1371/journal.pone.0038313

Figure Lengend Snippet: (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.

Article Snippet: The generation and validation of anti-PSD-95 mAb K28/43 (IgG2a) , anti-KChIP1 mAb K55/7 (IgG1) and anti-Kv4.2 mAb K57/1 (IgG1) , anti-Caspr/Paranodin mAb K65/35 (IgG1) , anti-Kv2.1 mAb K89/34 (IgG1) , anti-BK channel mAb L6/60 (IgG2a) were described previously. mAbs against QKI (N147/6, IgG2b), Ankyrin-G (N106/36, IgG2a) and GFAP (N206A/8, IgG1) were obtained from the UC Davis/NIH NeuroMab Facility, which also distributes K14/16, K28/43, K55/7, K57/1, K65/35, K89/34 and L6/60.

Techniques: Transfection, Immunofluorescence, Labeling, Expressing, Fluorescence, Western Blot, Recombinant, SDS-Gel, Molecular Weight

Journal: PLoS ONE

Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

doi: 10.1371/journal.pone.0038313

Figure Lengend Snippet: (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard & Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

Article Snippet: The generation and validation of anti-PSD-95 mAb K28/43 (IgG2a) , anti-KChIP1 mAb K55/7 (IgG1) and anti-Kv4.2 mAb K57/1 (IgG1) , anti-Caspr/Paranodin mAb K65/35 (IgG1) , anti-Kv2.1 mAb K89/34 (IgG1) , anti-BK channel mAb L6/60 (IgG2a) were described previously. mAbs against QKI (N147/6, IgG2b), Ankyrin-G (N106/36, IgG2a) and GFAP (N206A/8, IgG1) were obtained from the UC Davis/NIH NeuroMab Facility, which also distributes K14/16, K28/43, K55/7, K57/1, K65/35, K89/34 and L6/60.

Techniques: Transfection, Labeling, Fluorescence, Western Blot, Purification, SDS Page, Staining

Journal: PLoS ONE

Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

doi: 10.1371/journal.pone.0038313

Figure Lengend Snippet: Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.

Article Snippet: The generation and validation of anti-PSD-95 mAb K28/43 (IgG2a) , anti-KChIP1 mAb K55/7 (IgG1) and anti-Kv4.2 mAb K57/1 (IgG1) , anti-Caspr/Paranodin mAb K65/35 (IgG1) , anti-Kv2.1 mAb K89/34 (IgG1) , anti-BK channel mAb L6/60 (IgG2a) were described previously. mAbs against QKI (N147/6, IgG2b), Ankyrin-G (N106/36, IgG2a) and GFAP (N206A/8, IgG1) were obtained from the UC Davis/NIH NeuroMab Facility, which also distributes K14/16, K28/43, K55/7, K57/1, K65/35, K89/34 and L6/60.

Techniques: Knock-Out, Labeling