Panel A. TEM micrographs. The core, skin and glyco layers are indicated. The inset shows the presence of a lipid layer detached from the rest of the fibre. Bar corresponds to 100 nm. Panel B. AFM micrographs. The arrows indicate some of the cavities. The core, skin, glyco layers and an artificial halo are indicated. Lack of symmetry in the latter indicates that the halo is caused by the sectioning procedure and reflects material compressed and displaced by the knife as it approaches the hard skin layer. The bar corresponds to 500 nm. The height colour coding bar extends bottom to top from 0 to 100 nm. Panel C. Attachment strength of silk layers. Untreated fibres were successively ether extracted, vigorously washed in 0.1% Triton X-100 detergent solution and subjected to freeze-thaw cycles. After treatment cross-sections of fibres for each step were analyzed by TEM. The loss of layers from outside to inside is evident. The efficiencies for removal lipids, glycoproteins and the skin were 100%, 70% and 30%, respectively. The bar corresponds to 200 nm.

Filter strips obtained by western blotting and loaded with material extracted from the indicated fibre layers were stained with Ponceau S (Pon) or reacted with Concavalin A (ConA), pre-immune serum (PIS), S1Rx, S2Rx and S-pbs. The running position of a 200 kDa marker is indicated by the lines.

Panel A. Light Microscopy (LM) phase images of fibres treated with mixtures of different ratios between HAc and HCl. Bar corresponds to 4000 nm. Minor (mi, reinforcement) and major (ma, dragline) ampullate fibres are indicated. Panel B. Light Microscopy (LM) phase contrast images of dragline treated with LiSCN at the stated concentrations for 1 minute. Bar corresponds to 8000 nm. Panel C. Scanning Electron Microscope (SEM) images of dragline incubated for the indicated times with 99% Hfip. Bar corresponds to 1000 nm. Panel D. Video contrast images of draglines (left unstressed, right fractured by applying stress) soaked in 8 M urea and counterstained with Coomassie Brilliant Blue. Bar equals 4000 nm.

Dragline silk from N. clavipes was incubated with the indicated solvents and the soluble fraction was analysed by gel-electrophoresis followed by Coomassie staining (A) and western blotting employing antibodies S1Rx and enhanced luminescence (ECL) for detection. The molecular weights of marker proteins are indicated in kilo Daltons (kDa).

Panel A. Oil red staining of fibres. Silk fibres were treated with water (upper filament) or ether extracted (lower filament) followed by oil red staining. Bar corresponds to 2000 nm. Panel B. Concavalin A (Con A) staining of fibres. Fibres gently washed in phosphate buffered saline or vigorously washed in 0.1% Triton X-100 were reacted with biotinylated Con A. The presence of α-methyl mannoside (α-MM), an inhibitor of Con A, is indicated by “I”. Bound Con A was visualised by gold conjugated streptavidin and SEM. The bar equals 500 nm.

Panel A. Fluorescence labelling of silk fibres. Reactivities of Concavalin A (ConA), pre-immune serum (PIS), S-pbs, S1Rx and S2Rx to lesions of vigorously washed silk filaments. Phase contrast (Ph), immunfluorescence (IF) and merged images are shown. The bar indicates 4000 nm. Panel B. Immunogold staining of silk fibre cross-sections. Shown are transmission electron microscopy (TEM) images of dragline cross sections reacted with Concavalin A (ConA) or the indicated antibodies. The bar equals 200 nm.

Panel A. Cut end of a dragline incubated in 8 M urea. High resolution stereo image was acquired with a Zeiss Neofluar 100x/NA 1.3 oil immersion objective. A stereo lorgnette is required for obtaining the 3D effect. The upper arrows will appear as one arrow behind the object, whereas the lower arrow in the right picture will appear in front of it. They indicate the end of the conical shape pointing to the open ends. Fibrils can be seen in the central parts (lower arrow, left image) of the fibre. Panel B. Freeze fractured dragline incubated in 8 M urea. Image was acquired as in panel A. The fractured site is indicated by the lower arrows. The exposed interior shows that the fibre consists of many fibrils, which are twisted (upper arrow) and from bundles.

A dragline can be divided into a shell and a core with five major layers of different material composition (from exterior to interior): a lipid coat, a glyco coat, a skin, an outer core and inner core. The approximate relative extensions of each layer are indicated.