Methods related to protein biochemistry

2.1.11 Isolation of plant proteins for SDS-PAGE and Western Blot

20 to 30 mg of plant material (seedlings, leaves) was transferred into a 2 mL Eppendorf tube with 2 metallic beads, frozen in liquid nitrogen and smashed in a grinding mill (Retsch, MM2000)at an oscillating amplitude of 50 for 1 to 2 minutes. The plant powder was mixed with 1x PBS pH7.4 (1:10 diluted 10x PBS: 1,4 M NaCl (Roth), 27 mM KCl (Fluka), 10 mM Na2HPO4 (Roth), 2 mM KH2HPO4 (Fluka), pH 7.4) and vortexed briefly. The mix was transferred into a 1.5 mL Eppendorf tube and incubated on ice for 15 minutes. After this, the mix was then centrifuged two times at 14 000 rpm for 5 minutes to generate a clear supernatant. Proteins were either loaded directly or analysed by measurement of protein concentration in order to separate a defined amount of proteins.

2.1.12 Measurement of protein concentration

Measurement of protein concentration was mainly necessary for assessment of enzymatic activity assay and was done using Micro BCA^TM Protein Assay Kit from

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Pierce. A BSA standard was prepared with following concentrations: 0; 1; 2; 3; 5; 7.5;

10 and 15 µg BSA (Albumin fraction V, biotin free, Roth) in water up to a volume of 50 µL. The standard mix was transferred into a 95-well-plate and mixed with 50 µL of Micro BCA^TM Working reagent which was composed of 25 µL of Reagent MA, 24 µL of Reagent MB and 1 µL of Reagent MC. Sample proteins were diluted 1:50 in extraction buffer and 10 µL of diluted proteins were mixed with 40 µL of extraction buffer and 50 µL of Micro BCA^TM Working reagent. Incubation followed at 37 °C for 30 minutes. The reaction mix was analysis at 500 nm on an ELISA-reader (SLT spectra II, Zinsser Analytic GesmbH) and calculation of protein concentration was exhibited in MS Excel.

2.1.13 SDS-PAGE

Proteins were separated on a discontinous polyacrylamid gel prior to development by coomassie staining or western blot. Stacking and separation gel were mixed as given in Table 19.

compounds separation gel

(12.5 %)

stacking gel (5.7 %)

Acrylamide 30 % 3 x 833 µL 570 µL

Bisacrylamid 1 % 780 µL 390 µL

1,5 M Tris.HCl pH 8.8 (18.2 g Tris/100ml) ¹) 2 x 750 µL /

0.5 M Tris-HCl pH 6.8 (6 g Tris/100 mL) ²) / 750 µL

H2O 2 x 570 µL 2 x 640 µL

10 % SDS 60 µL 30 µL

10 % APS 36 µL 24 µL

TEMED 3,6 µL 2,4 µL

Total 10,06 mL 5,045 mL

Table 19: Compounds of separation and stacking gel for SDS‐PAGE.  

Acrylamine, SDS (sodium dodecyl sulfate) and TEMED (N,N,N’,N’‐Tetramethylethylenediamine) were from Roth; 

Bisacrylamid (Bis N,N’‐Methylene‐bis acrylamide) and APS (ammonium persulfate) from Biorad. ¹) separation gel  buffer, ²) stacking gel buffer. 

Preparation of polyacrylamid gels was done using BioRad System including gel preparation station and tank. After pouring of separation gel, isopropanol (Roth) was added to smooth the surface and facilitate polymerisation. Polymerisation followed for approximately 30 minutes. Isopropanol was removed and the separation gel was carefully rinsed with water. The stacking gel was poured, the comb was inserted and the gel was polymerised for 30 minutes.

The gel electrophoresis was performed with 1x running buffer (5x stock solution: 15 g/L Tris (Roth); 72 g/L glycin (Roth); 5 g/L SDS; water up to 600 mL, stored at + 4 °C;

for usage diluted 1:5). The proteins were mixed with equal volume of sample buffer (200 mg SDS (Roth), 5 mL stacking gel buffer pH 6.8 (see Table 19), 2.8 mL 87 % [w/v]

glycerol (Merck), 2.7 mL HQ-water, a few drops of bromphenolblue (Merck) and 3 mg/mL DTT (Dithiothreitol, Roth) added prior to use; stored at – 20 °C). Proteins mixed with sample buffer were incubated at 95 °C for 5 minutes and subsequently cooled on ice before loading. 4 µL of Fermentas PAGE RulerTM prestained protein ladder (SM0671) were loadeds. Separation followed at 0.01 A (100 V) per gel for approximately 1 ½ hours or at 200 V for 1 hour. The gel was then either stained with coomassie brilliant blue or further used for western blot.

2.1.14 Coomassie blue staining

The gel was incubated for 30 minutes in fixing solution (500 mL MetOH, 70 mL acetic acid and water up to 1 L) under shaking. The gel was then stained with coomassie brilliant blue G-250 (38 mL (3.5 %) of 60 % perchloric acid (Merck), 0.4 g (0.04 %) coomassie brilliant blue G-250 (BioRad) dissolved in 10 mL of methanol (Roth), water up to 1 L; mixed for 2 hours, filtrated) for 5 minutes on a shaker. Gel destaining followed with 5 % glacial acetic acid (Roth).

2.1.15 Western Blot

The SDS-PAGE gel sandwich was dissembled and the gel was incubated in 1x blotting buffer (10x blotting buffer stock: 250 mM Tris, 1.92 M glycin; 1x blotting buffer ready to use: 0.1 L 10x blotting buffer, 0.2 L methanol, 0.7 L water up to 1 L).

Nitrocellulose membrane (Poll Life Sciences, BioTraceTM NT, Pure Nitrocellulose Blotting Membrane) and Whatman paper (BioRad, extra thick filter paper 15x20 cm) was cut and also shortly incubated in blotting buffer. Semi-dry blotting was performed on a semi-dry blotter (Trans-Blot(R) SD Semi Dry Transfer cell, BioRad). The blotting sandwich was assembled according to the instructions. Both metal plates were prewet with blotting buffer. One prewet Whatman paper was placed on the bottom metal plate, followed by the nitrocellulose membrane. The gel was carefully placed on the membrane and covered with the second prewet Whatman paper. Air bubbles were removed with a wet glass tube. The top metal plate was placed on the blotting sandwich

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and locked. Finally the lid with both electrode junctions was put on. Blotting was done at 15 V for 25 minutes. After blotting, the nitrocellulose membrane was rinsed in 1x TBS-T (10x TBS stock, 1 L: 1 M Tris (Roth), 1 M NaCl (Roth), pH7.4; 1x TBS-T ready to use, 1 L: 10x TBS diluted 1:10, 0.05 % Tween-20). In some cases, ponceau staining was realised to check success of blotting. The membrane was covered with 0.5 % ponceau S (Sigma) in 1 % acetic acid and incubated for a few minutes. Ponceaus S was then removed and the membrane was carefully rinsed with water until protein bands were visible. The blot was washed with water and then blocked. Blocking was performed either with 1 - 3 % skimmed milk powder (Fixmilch instant) or with 0.5 % BSA (Roth) for 1 hour at room tempterature on a shaker or over night at + 4 °C. The blocked membrane was washed with 1x TBS-T two times for 10 - 15 minutes. Incubation with first antibody (pure or rabbit sera for testing, see Table 20) followed either at room temperature for 1 hour on a shaker or over night at + 4 °C. The antibody was diluted in 1x TBS-T according to the instructions. After that the membrane was washed twice with 1x TBS-T for 10 to 15 minutes and incubated with secondary antibody for 1 hour at room temperature on a shaker. The incubated membrane was washed in 1x TBS-T two times for 10 to 15 minutes and finally shortly in water prior to development. In most of the cases the secondary antibody used was conjugated with alkaline phosphatase (see Table 20) which enabled fast detection with BCIP/NBT from Sigma-Aldrich (colorimetric detection system). One tablet was dissolved in 10 mL of water in a 15 mL falcon tube covered in aluminium foil. The solution was placed directly on the membrane and incubated until bands were visible. The reaction was stopped by washing the membrane with water. In special cases, secondary antibodies conjugated with HRPO (see Table 20) were used and the detection followed by using Pierce SuperSignal West PICO Chemiluminescent Substrate (chemiluminiscent detection system). For that, the membrane was washed and placed on a plastic foil. A freshly prepared mix of detection solution I (luminol/enhancer) and detection solution II (stable peroxide buffer) mixed 1:1 (total 1 mL) were pipetted onto the membrane and dispersed by gentle shaking. The reaction was incubated for 1 minute and the membrane was then placed in a plastic foil and put into film cassette for development of the autoradiographic film (Amershan HyperfilmTM ECL, GE Healthcare). Exposure times ranged from a few seconds to several minutes. After development the membrane was washed, dried at room temperature and shrink-wrapped into plastic foil.

Primary antibody Secondary antibody detection of His-Tag present on

proteins expressed from vector p21Gt

 His*Tag Monoclonal Antibody, mouse IgG (70796-4, Novagen), 1:1.000 in 1x TBS-T + 0.5

% BSA

 Goat Anti-Mouse HRP

Conjugate (71045-3, Novagen),

1:5.000 in 1x TBS-T + 0.5 % BSA

detection of AtFUC95A

 sera against AtFUC95A produced in rabbits 1)

1:1.000 in 1x TBS-T + 3 % milk powder

 Affinity-purified AtFUC95A antibody

 Alkaline Phosphatase Anti- Rabbit IgG (H+L), Affinity purified, made in goat (AP- 1000, Vector Laboratories), 1:1.000 in 1x TBS-T + 3 % milk powder

 Anti-Rabbit IgG (whole

molecule) Peroxidase antibody produced in goat (A0545, Sigma),

Table 20: Overview about all primary and secondary antibodies used.  

1) Analysis of antibody against AtFUC95A produced in rabbits for potential usage in subcellular localisation studies. 

2) Antibodies used for detection of fucosyltransferases transiently expressed in N. benthamiana.  

2.1.16 Gel-affinity-chromatography

Affinity-chromatography has been performed in order to purify a specific antibody against AtFUC95A produces in rabbits. Purification has been performed using Affi-Gel 15 Gel from BioRad. The selected method was a modified version of anhydrous coupling of AtFUC95A peptide to the gel. First step was equilibration of the gel which was stored at – 20 °C. The gel was suspended before pipetting of 2 times 1 mL into two 2 mL Eppendorf tubes. The tubes were centrifuged at 800 rpm for 1 minute at room temperature and the supernatant was removed. This step was repeated until approximately 0.5 mL of solid gel was left in the 2 mL tube. The gel was mixed with water-free isopropanol and DMSO (Sigma-Aldrich, free of water by using a 3 Å molecular sieve) up to 2 mL and incubated for 3 to 4 minutes at room temperature. All supernatant was removed and coupling followed by mixing of 1.6 mL DMSO, 4 µL of triethylamine (Merck) and 1.5 µmol of dried peptide dissolved in DMSO with the gel and subsequent incubation over night at room temperature on a shaker rotating at low velocity. The gel was centrifuged at 800 rpm for 2 minutes and then mixed with 5 mL 1xTBS-T pH7.2 (0.1 M Tris-HCl pH7.2; 0.1 M NaCl; 2.5 mM MgCl2 (Roth); 0.05 % Tween-20). After incubation for 10 minutes under shaking, the gel was centrifuged again and the washing step was repeated. The gel pellet was mixed with 9 mL of 1xTBS-T pH7.2 and 1 mL of serum and incubated overnight at 4 °C under agitation.

The following day, the gel was centrifuged and the supernatant containing unbound

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peptide was collected. The gel was then blocked with 1.6 mL of water-free DMSO (Sigma-Aldrich), 5 µL of triethylamine (Merck) and 7.5 µL of butylamine (Merck) (primary amine to block unbound gel) and incubated for 4 to 6 hours at room temperature under shaking. After this the gel was transferred into a small column filled with 1xTBS-T and the flow-through was collected. The gel was washed with 5 mL of 1xTBS-T and the flow-through was collected. The elution was then done with 500 µL of 0.2 M glycine-HCl pH2.5 directly into 500 µL of neutralisation buffer (1 M Tris-HCl pH7.5). 10 fractions were collected in total. After elution the gel was washed with 1xTBS-T and the flow-through was also collected.

2.1.17 Fucosidase activity assay

Plant seedlings of AtFUC95A knockout line Fuc95A#13, AtFXG1 knockout line Atfxg1#27 and Col-0 wildtype or in N. benthamiana transiently expressed proteins were analysed by fucosidase activity assays. Seedlings from liquid culture were washed with deionised water and dried carefully. In case of transient expression in N. benthamiana, the infiltrated leaf area was cut out and smashed in 40 mM sodium acetate buffer at pH 5.0 (Léonard et al., 2008) at a ratio of 1 µL of buffer per mg of plant material. Sample preparation has been performed on ice using a cooled mortar and pestle. The smashed samples were centrifuged at 14 000 g for 15 minutes at 4 °C. The supernatant was collected and centrifuged. A volume of 4 µL of proteins and 4 µL of 80 pM substrate XXFG-PA (2-aminopyridine-labelled xyloglucans for analysis by HPLC (see 0) were mixed and incubated at 37 °C for 2 hours. The reaction was stopped at 95 °C for 5 minutes and analysis was performed by HPLC.

2.1.18 Fucosyltransferase 1 activity assay

Leaves were harvested two days after infiltration and ground on ice with a mortar in McIlvaine buffer (0.2 M Na2HPO4 * 12 H2O (Merck); 0.1 M Citric acid (C6H8O7 * H2O (Merck); pH adjusted with NaOH) (Ichinose et al., 2006) at pH 7.0 in a ration of 1 µL per mg leaf material. The crude extract was centrifuged two times at 14 000 rcf for 10 minutes and then directly used for the enzyme activity assay.

61.5 µL of supernatant were mixed with 10 µL of 10x protease inhibitor (complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets; Roche; 10x solution: 1 tablet in 1 mL of water), 2.5 µL of 0.5 µg/µL tamarind xyloglucan (Sigma), 15 µL of 100 mM GDP-

L-fucose, 10 µL of 10 % Triton X-100 and 1 µL of 1 M MnCl2 to give final 100 µL.

Incubation followed at 37 °C for 2 hours and 24 hours. After incubation, the enzyme reaction was stopped by freezing at – 80 °C prior to digestion with cellulase in order to release xyloglucan oligosaccharides. To release xyloglucan oligosaccharides, 0.1 mg of cellulase (Cellulase “Onozuka” from Trichoderma viride, 1 U/mg, Serva) was dissolved in 10 mL of 200 mM sodium acetate buffer pH 4.5. 900 µL of this solution were mixed with the enzyme reaction mix and incubated at 37 °C for 3 hours. Purification followed using carbon columns (2.1.26). Analysis followed by ESI-TOF-MS.

2.1.19 Acetylesterase activity assay

AtFXG1, AtFUC95A and the empty vector control ppT8 were transiently expressed in N. benthamiana leaves. Leaves were collected two days later and smashed in 40 mM Tris.HCl buffer pH 7.7 at a ratio of 1 µL buffer per mg leaf material. Samples were ground on ice in a mortar and then two times centrifuged for 15 minutes at 14 000 g at 4

°C. Protein concentration was measured using Micro BCA Protein Assay Kit from Pierce and the protein samples were diluted to 3 µg/µL.

The substrate used was pNP-acetate from Sigma. An amount of 50 mg of pNP- acetate were solubilised in 500 µL of MetOH until a saturated solution was obtained.

This solution was shortly centrifuged and the supernatant was transferred under strong agitation into 10 mL of water and shortly centrifuged again. The acetylesterase activity assay was prepared by mixing of 100 µL of saturated pNP -acetate solution, 20 µL of buffer 40 mM Tris.HCl pH 7.7 and 5 µL of sample proteins. Incubation followed at room temperature for several time points ranging in 20 minutes intervals until final 2 hours.

Measurement was performed at 405 nm using an ELISA-reader (SLT spectra II, Zinsser Analytic GesmbH).

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