For immunoblotting of AU-PAGE, an equivalent of 1.5M sperm of pooled basic protein extraction was used. The proteins were transferred towards the negative pole onto a PVDF membrane (pore size 0.45μm; Roth, Karlsruhe Germany), blocked and incubated with the primary antibody overnight (antibody dilutions shown in S3 Table ). For SDS PAGE, proteins were transferred towards the positive pole onto a PVDF membrane (pore size 0.45μm; Roth, Karlsruhe Germany). The membranes were then washed and incubated with an HRP-labelled secondary antibody followed by chemiluminescent detection using Westar NOVA 2.0 chemiluminescent substrate (Cyanagen, Bologna, Italy) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, USA). Since Merges et al. [38 (link)] was able to show that (outer dense fiber protein 2) ODF2, which is a major component of the sperm tail, is compatible with AU-PAGE and represents one of the prominent bands in the upper part of the Coomassie AU gel we used the protein as a loading control.
Pvdf membrane
Manufactured by Carl Roth
Sourced in Germany
PVDF membrane is a type of polymer-based membrane made from polyvinylidene fluoride (PVDF). It is a durable and chemically resistant material commonly used in various laboratory and industrial applications. The core function of PVDF membrane is to act as a filtration or separation medium, allowing the selective passage of certain molecules or particles while retaining others, depending on the specific pore size and properties of the membrane.
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Lab products found in correlation
Chemidoc touch imaging system, by Bio-Rad (9 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Ecl plus kit, by GE Healthcare (3 mentions)
Nupage sds gel, by Thermo Fisher Scientific (3 mentions)
Nanovue plus spectrophotometer, by GE Healthcare (3 mentions)
Ecl western blotting analysis system, by Cytiva (3 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor, by Roche (3 mentions)
Nativepage 5 g 250 sample additive, by Thermo Fisher Scientific (2 mentions)
Native page 3 12 bis tris, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ecl western blotting analysis system, by GE Healthcare (2 mentions)
Sonoplus, by Roche (2 mentions)
Image lab 5, by Bio-Rad (2 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Microtube, by Sarstedt (2 mentions)
85 protocols using pvdf membrane
1
Immunoblotting of Basic Sperm Proteins
2
Evaluation of Adenylyl Cyclase 1 Expression
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Tissue samples of control and pilocarpine-treated animals containing only the subiculum were homogenized at 4°C in extraction buffer [150 mM NaCl, 50 mM Tris, 10 mM HEPES, 1% Triton X-100, 1% EGEPAL, proteinase inhibitor cocktail (Roche)]. Homogenates were centrifuged at 20,000 g for 15 min and the resulting supernatant was used for SDS-PAGE and protein determinations with the BCA assay (Pierce, Rockford, IL, USA). Western blot analysis was performed using 5–20% gradient SDS-PAGEs. Gels were electro blotted on PVDF membranes (Roth, Karlsruhe, Germany) for 2 h and membranes subsequently blocked for 1 h in TBST buffer (100 mM Tris-HCl; 0.9% NaCl, 1% Tween 20, pH 7.4) containing 5% non-fat dry milk. Blots were incubated overnight at 4°C with specific primary antibodies (anti AC-1 1:200, SC-25743, Santa Cruz Biotechnology, Santa Cruz, CA, USA, anti-Actin 1:5,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and immunoreactivity was visualized using HRP-coupled goat anti-rabbit or goat anti-mouse secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Quantification of Western blots was done by densitometric analysis using the NIH Image program v1.61. The relative expression levels of adenylyl cyclase 1 (AC1) and actin as well as the ratio of AC1 and actin are expressed as mean values ± standard error of mean (SEM).
3
Protein Detection via SDS-PAGE and Western Blot
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Where described, protein samples were labeled with lightning red (Serva) diluted 1:50 in protein sample buffer and then separated on anyKD (Biorad) or 12.5% SDS-PAGE gels. Subsequently, total protein staining was analyzed using a Typhoon trio (GE Healthcare). After transfer to PVDF membranes (Carl Roth), membranes were blocked with 5% skimmed milk/TBST, incubated with primary antibodies overnight at 4 °C, followed by washing with TBST and incubation with HRP-conjugated second step antibodies for one hour. Luminescence was detected using ECL reagent and x-ray films (GE Healthcare). Films were scanned and where mentioned, densitometric quantification was performed using ImageJ.
4
Mitochondrial Protein Complex Analysis
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75 μg of mitochondria were pelleted by centrifugation and resuspended in BN-lysis buffer (Wittig et al., 2006 (link)). For analysis of F1FO-ATP synthase or MICOS complex 1 % (w/v) or 3 % (w/v) digitonin were used. Cleared lysates were supplemented with Native PAGE 5% G-250 Sample Additive and subjected to BN-PAGE (Native PAGE 3–12% Bis-Tris; Life Technologies, Carlsbad, CA, USA). After blotting on PVDF membranes (Roth, Karlsruhe, Germany) immuno-decoration using the indicated antibodies was performed.
5
Western Blot Analysis of CagA Protein
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Fifty micrograms of whole cell lysates were loaded on 8–10% polyacrylamide gels and separated by SDS PAGE. Proteins were transferred onto nitrocellulose or PVDF membranes (Carl Roth, Karlsruhe, Germany) using semidry transfer blotting. Membranes were blocked in 3% bovine serum albumin (BSA) supplemented with 0.05 mM Na3VO4 and incubated with primary antibodies overnight at 4 °C. CagA was analyzed using polyclonal anti-CagA antibodies detecting the N-terminal region of CagA (Paul-Ehrlich Institute, Langen, Germany) [16 (link)] or the C-terminal region (raised against amino acids 890-1186) [15 (link)]. Phosphorylated CagA was detected using the anti-phosphotyrosine antibody 4G10 (gift from Prof. Stephan Feller, Halle an der Saale, Germany), anti-GAPDH antibody recognized GAPDH (Cell Signaling, Frankfurt am Main, Germany) and anti-HtrA antibody raised against Hp HtrA (Paul-Ehrlich Institute, Langen, Germany) [28 (link)]. Densitometric analysis of Western blots was performed from at least three to four independent experiments. For quantification, band intensities were measured with Image Lab software (version 6.0.1, Bio-Rad, Vienna, Austria) and normalized to GAPDH. Statistics were analyzed with one-way ANOVA followed by Tukey’s test for multiple comparison correction.
6
Total Protein Extraction and Western Blot Analysis
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For total protein extraction, plant material was grinded and incubated with an extraction buffer composed of 50 mM Na‐phosphate pH 7.4, 150 mM NaCl, 10% (v/v) glycerol, 5 mM EDTA, 0.1% (v/v) Triton X‐100, 1 mM DTT, 2 mM Na3VO4, 2 mM NaF, 1% (v/v) Protease Inhibitor Cocktail (Sigma), and 50 μM MG132, as previously described (Arongaus et al, 2018 ).
Proteins were separated by electrophoresis in 8% (w/v) SDS–polyacrylamide gels and transferred to PVDF membranes (Roth) according to the manufacturer's instructions (iBlot dry blotting system, Thermo Fisher Scientific), except for CRY2 immunoblots, which were transferred on nitrocellulose membranes (Bio‐Rad).
For protein gel blot analyses, anti‐UVR8(426–440) (Favory et al,2009 ), anti‐UVR8(1–15) (Yin et al, 2015 ), anti‐UVR8(410–424) (Heijde & Ulm, 2013 ), anti‐GFP (Living Colors® A.v. Monoclonal Antibody, JL‐8; Clontech), anti‐actin (A0480; Sigma‐Aldrich), and anti‐CRY2(588–602) (Eurogentec, raised against the peptide N′‐CEGKNLEGIQDSSDQI‐C′ and affinity purified) were used as primary antibodies. Horseradish peroxidase‐conjugated anti‐rabbit and anti‐mouse immunoglobulins (Dako) were used as secondary antibodies. Signal detection was performed using the ECL Select Western Blotting Detection Reagent (GE Healthcare) and an Amersham Imager 680 camera system (GE Healthcare).
Proteins were separated by electrophoresis in 8% (w/v) SDS–polyacrylamide gels and transferred to PVDF membranes (Roth) according to the manufacturer's instructions (iBlot dry blotting system, Thermo Fisher Scientific), except for CRY2 immunoblots, which were transferred on nitrocellulose membranes (Bio‐Rad).
For protein gel blot analyses, anti‐UVR8(426–440) (Favory et al,
7
Western Blot Analysis of Protein Expression
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Cells were lysed in lysis buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM NaF, 2 mM Na3VO4, 100 mM PMSF, 5 µg/mL Aprotinin, 5 µg/mL Leupeptin, and 3 µg/mL Pepstatin A. Protein was separated by SDS-PAGE under reducing conditions and transferred onto PVDF membranes (Roth, Karlsruhe, Germany). Blots were blocked in TBS buffer containing 0.05% Tween 20 and 5% non-fat dry milk for 1 h at room temperature. The membrane was incubated overnight at 4 °C with the respective primary antibodies. The secondary antibody was incubated for 1 h at room temperature. Detection of antibody binding was performed by enhanced chemiluminescence (ECL Western blotting analysis system, GE Healthcare, Freiburg, Germany). Equal loading was verified by detecting β-actin or tubulin. Where indicated, protein levels were quantified by densitometry using ImageJ software (ImageJ 1.40 g NIH, Madison, WI, USA). All Western blot experiments were repeated at least once in independent experiments.
8
Immunoblotting Analysis of Cellular Proteins
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Cultured cells were harvested using a cell scraper and washed three times with 1xPBS. Cell pellets were disrupted in solution A containing phosphatase inhibitors (Sigma-Aldrich) by sonication. Cell debris were removed by centrifugation at 1000 g and 4 °C for 10 min and the supernatant was collected. Protein concentration was determined using BioRad protein assay. Twenty μg protein were separated by SDS-PAGE and blotted onto PVDF membranes (Carl Roth). Proteins were detected by using the antibodies: GAPDH (Cat# 2118S), ATGL (Cat# 2138S), HSL (Cat#4107), mTOR (Cat# 2972), P-mTOR (Cat# 2974S), P-S6K (Cat# 9205), Bcl-XL (Cat# 2762), Bcl-2 (Cat# 2876S), AMPK and ACC Antibody Sampler Kit (Cat# 9957) from Cell Signaling (Massachusetts, USA), MGL (100035) from Cayman Chemical (Michigan, USA) and respective horseradish peroxidase conjugated secondary antibodies (A120-201P, Bethyl laboratories Inc., Texas, USA). Signal densities were analyzed using BioRad ChemiDoc MP system.
9
Protein Expression Analysis by Western Blot
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Cells were lysed in 200 μl lysis buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM NaF, 2 mM Na3VO4, 100 mM PMSF, 5 μg/ml Aprotinin, 5 μg/ml Leupeptin, and 3 μg/ml Pepstatin A. Protein was separated by SDS-PAGE under reducing conditions and transferred onto PVDF membranes (Roth, Karlsruhe, Germany). Blots were blocked in TBS buffer containing 0.05% Tween 20 and 5% non-fat dry milk for 1 h at room temperature. The membrane was incubated overnight at 4 °C with the respective primary antibodies. The secondary antibody was incubated for 1 h at room temperature. Detection of antibody binding was performed by enhanced chemoluminescence (ECL Western blotting analysis system from GE Healthcare, Freiburg, Germany). Equal loading was verified by antibodies against β-actin or Tubulin. Where indicated protein levels were quantified by densitometry using ImageJ software (ImageJ 1.40 g NIH, USA). At least two independent western blot experiments were performed.
10
Quantifying Protein Abundance by Western Blot
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Total protein was isolated from 200 mg of seedlings with Laemmli Buffer. Total proteins weighing 15 µg were separated by 12% SDS-PAGE and transferred to polyvinyl difluoride (PVDF) membranes (Roth, Karlsruhe, Germany) by wet blotting at 4°C in 25 mM Tris-HCl at pH 8.3, 24 mM glycine and 10% ethanol for 2 h at 250 mA. Enhanced chemiluminescence detection was performed using an ECL Western Blotting Substrate kit as recommended by the manufacturer (Thermo Scientific, Rockford, USA). Mouse anti-GFP antibodies [632381(JL-8), Clontech], at a dilution of 1:1000, were used for immunoblotting. Secondary antibodies were Alexa Fluor® 555 goat anti-mouse (Fig. 5A ) SFX Kit (A31621, Invitrogen Life Technologies) and goat anti-mouse HRP (Fig. 5B ) (170–6516, Biorad, Solna, Sweden). Western blots were performed independently three times and gave consistent results; one representative blot is shown.
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