An antibody-antigen complex was prepared by adding 100 µg of protein lysate and primary antibody in RIPA buffer to a total volume of 1 ml, then incubated overnight at 4 °C on a rotary tube mixer. The following day, 50 µl of protein-G magnetic beads (Millipore, LSKMAGG10) were dispensed into a 1.5 ml collection tube and placed on a magnetic rack (Millipore, 20–400). The storage substrate was removed and the beads were washed twice in 500 µl PBS with 0.1% Tween-20 (PBST). The mixture was briefly vortexed and centrifuged between washes and the supernatant was discarded. Next, the antibody-antigen complex was mixed with the magnetic beads and incubated overnight on a rotary tube mixer at 4 °C. The next day, the mixture was briefly centrifuged and washed 3 times in PBST. The elution was resuspended in 25 µl RIPA buffer with reducing agent (ThermoFisher, NP0004) and sample buffer (ThermoFisher, NP0007). Next, the samples were denatured by heating at 95 °C for 10 and loaded into 4–12% Bis-Tris pre-cast protein gels for SDS-PAGE.
Lskmagg10
Manufactured by Merck Group
The LSKMAGG10 is a piece of laboratory equipment manufactured by Merck Group. It is designed for general laboratory use, but a detailed description of its core function is not available at this time.
Automatically generated - may contain errors
5 protocols using lskmagg10
1
Antibody-Antigen Complex Purification
2
Arl4C Interactome Analysis in HeLa Cells
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HeLa cells in 10-cm dishes were transiently transfected with 6 µg pSG5-Arl4C WT, 7.2 µg pSG5-Arl4C Q72L, 15 µg pSG5-Arl4C T44N, or 15 µg pSG5 vector using Lipofectamine 2000 transfection reagent. After 16 h of transfection, the cells were lysed with immunoprecipitation (IP) buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40; 1× protease inhibitor cocktail); the lysate was then centrifuged for 10 min at 14,000 × g at 4°C. The supernatants were incubated with protein G magnetic beads (catalogue: LSKMAGG10; Millipore) conjugated with 1 µl of anti-FLNa antibody for 3 h at 4°C using end-over-end agitation and then washed five times with IP buffer. The coimmunoprecipitated proteins were boiled after adding 30 µl 4× protein-loading buffer and analyzed by immunoblotting.
3
Immunoprecipitation Protocol for Protein Analysis
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RIPA (20 mmol/L HEPES pH 7.5, 150 mmol/L NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol) extracts were pre-cleared using 10 μg/mL of pre-immune rabbit or mouse immunoglobulin G (Millipore, Burlington, MA) for 2 hours at 4°C. One μL of protein G magnetic beads (Millipore; LSKMAGG10) per 10 μg of antibody was added, and the sample was incubated for another 1 hour at 4°C. After removal of beads, proteins were immunoprecipitated using 10 μg/mL of the IP antibody overnight at 4°C. Ten μL of protein G magnetic beads per 1 μg of antibody was added, and the sample incubated for 4 hours at 4°C. The beads were magnetically separated and washed 2 times in RIPA buffer. Beads were then resuspended in 10 μL of 4x sodium dodecyl sulfate loading buffer (0.25 mol/L Tris pH 6.8, 40% glycerol, 20% β-mercaptoethanol, 4% sodium dodecyl sulfate), and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
4
Quantifying Phosphorylated Cystathionine Synthase
5
Immunoprecipitation and Mass Spectrometry
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Cells were washed with ice-cold PBS and lysed in 500 μL coIP buffer (150 mM NaCl, 50 mM pH 7.4 Tris-HCl, 0.5% NP-40, 1 mM EDTA, 5% glycerin and protease inhibitors). The lysates were sonicated for 8 × 5 s with a 10 s pause on ice. Then the lysates were clarified by centrifugation at 12,000 ×g for 15 min, and the supernatant was passed through a 0.45 μm membrane syringe filter. 40 μL of the supernatant was taken as input, and the rest was incubated with indicated primary antibody overnight at 4°C. Then 10 μL protein A (Millipore, LSKMAGA10) and protein G (Millipore, LSKMAGG10) magnetic beads were added to the antibody-protein complexes and incubated at 4°C for 2h. After the beads were washed 6 times with coIP buffer, the protein complexes were collected by 2×SDS-PAGE sample buffer and analyzed by western blot. For mass spectrometry, samples were processed by Novogene's proteomics & mass spectrometry facility.
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