One hundred picomoles of various lipids was spotted in a nitrocellulose membrane (Lipid Strips; Echelon Biosciences). The membranes were blocked with 2% skim milk in phosphate-buffered saline (pH 7.4) for 1 h at 4 °C. After blocking, 10 ml of 3% fatty acid–free bovine serum albumin and 0.1% Tween 20 in phosphate-buffered saline (pH 7.4) containing 6× His-tagged HSP27 (final concentration: 20 nM) was added to the membranes. The membrane was incubated for 20 min at 4 °C and was then incubated with an anti-6× His antibody for 1 h at 4 °C, followed by incubation with anti-mouse IgG conjugated with horseradish peroxidase (Bethyl Laboratories, Montgomery, TX, USA) antibody. Finally, lipid-bound 6× His-HSP27 was visualized using an enhanced chemiluminescence Western blotting detection system (GE Healthcare, Little Chalfont, UK).
Lipid strips
Manufactured by Echelon Biosciences
Lipid strips are a type of analytical laboratory equipment used to separate and identify different lipid species within a sample. They consist of a solid support material, typically a thin layer of silica gel, onto which a mixture of lipids can be applied. Through the process of thin-layer chromatography, the lipids are separated based on their relative affinities for the solid support and the mobile phase, allowing for the visualization and quantification of individual lipid components.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence western blotting detection system, by GE Healthcare (1 mentions)
Anti mouse igg conjugated with horseradish peroxidase, by Fortis Life Sciences (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Anti flag m2 agarose, by Merck Group (1 mentions)
Pip array, by Echelon Biosciences (1 mentions)
Adp glotm kinase assay kit, by Promega (1 mentions)
Tr bsa, by Merck Group (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated egf, by Thermo Fisher Scientific (1 mentions)
Mbp rabbit polyclonal antibody, by Merck Group (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Anti his hrp antibody, by Merck Group (1 mentions)
Anti gst antibody, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
11 protocols using lipid strips
1
Lipid Binding Assay for HSP27
2
Protein-lipid Binding Assay Protocol
3
PISD Lipid Binding Assay
4
Lipid Binding Assay for FadD13
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Lipid strips were purchased
from Echelon.
The strips were washed with 4 × 5 mL of wash buffer [10 mM phosphate
buffer (pH 7.4), 2.7 mM potassium chloride, 137 mM sodium chloride,
0.05% Tween 20, and 0.5% BSA] and blocked with 5 mL of blocking buffer
[10 mM phosphate buffer (pH 7.4), 2.7 mM potassium chloride, 137 mM
sodium chloride, 0.05% Tween 20, and 1% BSA] for 1 h at room temperature.
The strips were washed, 4 × 5 mL for 5 min, and thereafter incubated
with a 6 μM protein solution (FadD13 in wash buffer) for 1 h
at room temperature. The strips were subsequently washed, 4 ×
5 mL for 5 min, and incubated with the primary antibody (mouse anti-His)
diluted 1:2000 in wash buffer for 1 h at room temperature. The strips
were washed again, 4 × 5 mL for 5 min, and incubated with a secondary
antibody (donkey anti-mouse) and diluted 1:2000 for 1 h at room temperature.
Afterward, the strips were washed, 4 × 5 mL for 5 min, and detected
with 2 mL of a TMB solution.
from Echelon.
The strips were washed with 4 × 5 mL of wash buffer [10 mM phosphate
buffer (pH 7.4), 2.7 mM potassium chloride, 137 mM sodium chloride,
0.05% Tween 20, and 0.5% BSA] and blocked with 5 mL of blocking buffer
[10 mM phosphate buffer (pH 7.4), 2.7 mM potassium chloride, 137 mM
sodium chloride, 0.05% Tween 20, and 1% BSA] for 1 h at room temperature.
The strips were washed, 4 × 5 mL for 5 min, and thereafter incubated
with a 6 μM protein solution (FadD13 in wash buffer) for 1 h
at room temperature. The strips were subsequently washed, 4 ×
5 mL for 5 min, and incubated with the primary antibody (mouse anti-His)
diluted 1:2000 in wash buffer for 1 h at room temperature. The strips
were washed again, 4 × 5 mL for 5 min, and incubated with a secondary
antibody (donkey anti-mouse) and diluted 1:2000 for 1 h at room temperature.
Afterward, the strips were washed, 4 × 5 mL for 5 min, and detected
with 2 mL of a TMB solution.
5
Purification and Analysis of GST-tagged Cdr2-Cter
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Expression of GST–Cdr2-Cter or GST was induced in BL21 bacteria from the pGEX6p-1–derived plasmid described in Production of mutant and tagged strains. In brief, cells were grown overnight in LB (Luria–Bertani) medium supplemented with 100 µg/ml ampicillin at 37°C. 500 ml LB and 100 µg/ml ampicillin were inoculated with 12.5 ml of the saturated culture, grown for 4 h at 30°C. Protein expression was induced by the addition of 1 mM IPTG and incubation for 1 h. Bacterial pellets were resuspended in 5 ml PBS, digested with 1 mg/ml lysozyme, treated with 1 µg/ml DNase I, sonicated twice for 1 min (50% amplitude), incubated with 1% Triton X-100 in PBS buffer at 4°C, and centrifuged 15 min at 4°C at 10,000 g. Soluble extract was incubated with 200 µl glutathione–Sepharose beads at 50% slurry for 2 h at 4°C. Finally, beads were washed 3× with cold PBS and eluted in four steps in 100 µl elution buffer (15 mM reduced glutathione and 50 mM Tris-HCl, pH 8). Eluted proteins were incubated with lipid strips (Echelon, Inc.) following the manufacturer’s protocol. lipid strips were probed with a rabbit affinity-purified anti-GST antibody (1:100; gift from J. Dumont).
6
Characterizing MYO1D Lipid Binding
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HEK293T cells (ATCC CRL-11268, authenticated and used between passages 5-10) overexpressing human FLAG-MYO1D were lysed in Nonidet P-40 buffer [50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1% (vol/vol) Nonidet P-40, 10% (vol/vol) glycerol, 1.5 mM EDTA and Protease Inhibitor Mixture]. Immunoprecipitation was performed using anti-FLAG M2 agarose (Sigma-Aldrich). Binding of purified FLAG-MYO1D to lipid was analyzed using commercially available lipid strips (Echelon Biosciences) according to the manufacturer's instructions.
7
Lipid Binding Assay for LtpM
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Phospholipid overlay assays were performed with lipid strips and PIP arrays purchased from Echelon Biosciences (Salt Lake City, UT). Nitrocellulose membranes pre-spotted with different phospholipids were blocked with 3% fat-free BSA in 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1% Tween 20 for 1 h at room temperature and incubated with 100 nm recombinant LtpM constructs in 2 ml of blocking buffer at 4 °C for 5 h. Binding of the proteins to lipids was visualized by immunodetection with primary anti-LtpM mouse polyclonal antibody and secondary anti-mouse IgG antibody (Table 3 ).
8
Antibody Sources and Reagents for Cellular Assays
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Beclin1 rabbit polyclonal antibody was purchased from Medical & Biological Laboratories; EEA1 and RAB5 mouse monoclonal antibodies from BD Bioscience; EEA1, Vps34, Beclin1, and Lamp1 rabbit monoclonal antibodies from Cell Signaling Technologies; EGFR rabbit polyclonal antibody from Abcam; PtdIns3P mouse monoclonal antibody from Echelon Biosciences; and Flag and Myc mouse monoclonal antibodies and MBP rabbit polyclonal antibody from Sigma-Aldrich. LMP-1 mouse polyclonal antibody was a gift from X. Wang. WDR81 antibody was generated in guinea pigs and rabbits by injecting purified GST-WDR81(332-604). WDR91 antibody was generated in mouse by injecting GST-WDR91(406-730). BEC-1 antibody was generated in rabbits or mice by injecting purified GST-BEC-1. GFP antibody was generated in mouse and rabbits by injecting purified His-GFP. Alexa Fluor dye–conjugated secondary antibodies, LysoTracker red DND-99, Dextran blue (MW 10,000), and Alexa Fluor 647–conjugated EGF were purchased from Life Technologies; TR-BSA from Sigma-Aldrich; ADP-GloTM Kinase Assay kit from Promega; and lipid strips from Echelon Biosciences.
9
GSDMA Lipid Binding Assay
10
Lipid-Binding Protein Interaction Assay
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Lipid strips (Echelon Biosciences) were blocked at room temperature for 1 h in TBST (50 mM Tris pH 7.5, 3 mM KCl, 137 mM NaCl, 0.1% Tween 20) containing 3% BSA and then incubated overnight with 300 pmol His-tagged NttA. The membrane was washed three times with blocking buffer and then incubated for 2 h at room temperature in the same buffer containing anti-His-HRP antibody (Sigma) diluted 1:2,000 and then treated with enhanced chemiluminescence substrate (ECL; Pierce) before detection by enhanced chemiluminescence.
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