- 711079 - Invitrogen Antibodies SERPINA1 antibody

Frey AM, Chaput D, Shaw LN
Cell reports 2021 Apr 6;35(1):108930

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Whole cell extract (30 µg lysate) of HT-29 (Lane 1) and HT-29 condition media (Lane 2). The blots were probed with Anti-alpha-1 antiTrypsin/A1AT Recombinant Rabbit Polyclonal Antibody (Product # 711079, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 55 kDa band corresponding to alpha-1 antiTrypsin/A1AT was observed in both cell lysate and condition media tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of A1AT was achieved by transfecting HT29 cells with A1AT specific siRNA (Silencer® select Cat # s10458 and s10459). Western blot analysis (Fig a) was performed using Whole cell extract from the A1AT knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-A1AT Recombinant Rabbit Polyclonal Antibody (Product # 711079, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to A1AT.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis, Hep G2 cells were fixed and permeabilized for detection of endogenous alpha-1 antiTrypsin/A1AT using Anti-alpha-1 antiTrypsin/A1AT Recombinant Rabbit Polyclonal Antibody (Product # 711079, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of alpha-1 antiTrypsin (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating golgi localization of alpha-1 antiTrypsin. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. Validation of V8 serum substrates identified by N-terminomics Serum was treated with varying concentrations of V8. Linear schematics depict the primary structure of proteins, with domains from UniProt/UniRef indicated. Cleavage sites inferred by N-terminomics are indicated by red markers, the scissile bond/V8 cleavage site is indicated by an asterisk with surrounding residues shown. Fragments and their ~predicted mass (calculated using ProtParam), with degradation products visible in corresponding western blots, are shown on schematics for serotransferrin, alpha-2-macroglobulin, SERPINA1, and SERPINC1. Prothrombin produced no visible degradation products; instead, the intact form and native products (produced by proteolytic activation) are depicted and visible in the untreated serum blot. AP1, activation peptide fragment 1; AP2, activation peptide fragment 2; light, thrombin light chain; heavy, thrombin heavy chain; TF1, Transferrin-like domain 1; TF2, transferrin-like domain 2; Ig, Ig-like fold; BR, bait region; RBD, receptor binding domain; RCL, reactive center loop. See also Figure S4 .

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