HEK293T cells were infected with ASFV-P31 at an MOI of 1 for the co-IP assay. At 48 hpi, the cells were lysed with NP-40 for 30 min at 4°C and centrifuged for 10 min at 4°C; the cellular supernatants were incubated with an irrelevant isotype antibody IgG (catalog no. A7016; Beyotime) serving as a control and anti-H240R PAb with protein G plus agarose (catalog no. IP10-10ML; Merck-Millipore) at 4°C for 12 h. The agarose was washed six times with phosphate-buffered saline (PBS), and the bound proteins were subjected to Western blotting (49 (link)).
Protein g plus agarose
Manufactured by Merck Group
Protein G Plus Agarose is a laboratory product used for the purification and isolation of immunoglobulins from various biological samples. It consists of protein G, a bacterial surface protein, covalently coupled to agarose beads. Protein G has a high affinity for the Fc region of immunoglobulins, allowing for the efficient capture and separation of these molecules.
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Lab products found in correlation
Hdac6, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Criterion tris hcl gel, by Bio-Rad (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
5 protocols using protein g plus agarose
1
ASFV-P31 Co-Immunoprecipitation Assay
2
Immunoprecipitation and Western Blot Analysis
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Cells were lysed on ice for 20 min in a buffer containing 1% Triton X-100, 10 mM Tris (pH 7.6), 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 mM EDTA, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Roche). The resulting lysates were centrifuged at 16,000 × g at 4 °C, and the supernatant fraction was incubated with antibodies against FLAG (Cell Signaling Technology), and HDAC6 (Cell Signaling Technology) for 2 h at 4 °C, followed by incubation with Protein G Plus Agarose (Merck Millipore). Immunoprecipitants were washed two times with the lysis buffer, boiled in 5× SDS loading buffer for 10 min, followed by separation on SDS-PAGE and analysis by western blot. Western blot analysis was performed as described above. For inflammatory IL-1β-induced protein interaction, WT or Parkin−/− OCPs were cultured for 3 days with 10 ng/ml of IL-1β in the presence of RANKL and M-CSF. Then, the anti-HDAC6 immunoprecipitation was performed.
3
Co-immunoprecipitation of AR and Myc-tagged proteins
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COS cells were transfected with mARVa-myc or mARVc-myc together with Rat AR-FL using Lipofectamine 2000 (Life technologies) as per the manufacture’s protocol. About 48 hrs. post-transfection cells were lysed with 1% CHAPS lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2 supplemented with protease inhibitor cocktail (Pierce). Then 500 μl cleared cell lysate were immunoprecipitated by incubation with 5 μg anti-myc mAb (Roche) and Protein G PLUS-Agarose (EMD Millipore) overnight at 4°C. The isotype matched control mAbs were included in all immunoprecipitation experiments as negative control. Immunoprecipitates were washed twice in 0.1% lysis buffer followed by elution separation on 4–20% Criterion Tris-HCl Gel (Bio-Rad) under reduced conditions.
4
Affinity Purification of Shp2 Complex
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A431 cells were transduced with lentiviral RapR-Shp2-mVenus and mCherry-FRB adenovirus, lentiviral CA-Shp2-mVenus, or mCherry-FRB adenovirus. Confluent, stably expressing, A431 cells were activated with 1µM rapamycin for 2 h. Protein G Plus Agarose (IP04; Sigma) beads were preincubated with anti-GFP antibody for 1 h in lysis buffer. Samples were lysed with HEPES sample buffer (20 mM HEPES-KOH pH 7.8, 50 mM KCl, 1 mM EGTA, and 1% NP-40 [vol/vol]) and centrifuged at 12,000 rpm for 10 min. Lysate was incubated with anti-GFP conjugated Protein G Plus beads for 1.5 h at 4°C. Following incubation, beads were washed twice with 1 ml of lysis buffer and twice with 1 ml of wash buffer (20 mM HEPES-KOH, pH 7.8, 250 mM NaCl, 50 mM KCl, 1 mM EGTA, 1% NP-40 [vol/vol]). Proteins were eluted with Laemmli sample buffer and resolved via SDS/PAGE and immunoblotting.
5
MIST1 Regulation of SNAI1 Expression
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Direct combination and transcriptional activation of SNAI1 by MIST1 was predicted by the Encyclopedia of DNA Elements in the UCSC database (http://genome.ucsc.edu/ENCODE/ ). The JASPAR database (http://jaspar.genereg.net/ ) was used to predict the MIST1 binding motif. Cells were fixed with 1% formaldehyde and terminated with 2.5 mM glycine. The scraped cells were sonicated for lysis in PBS with sodium thiosulfate. The lysates were divided into three aliquots, one of which was a positive control and received no treatment and one of which was a negative control, which was incubated with immunoglobulin G (cat. no. I5006) and protein G PLUS-Agarose (cat no. P7700) 9both from Sigma-Aldrich; Merck KGaA). One third of the cell lysate was used as the test group, and was incubated with antibody against MIST1 (1:100) and protein G PLUS-Agarose. After removal of RNA and protein, DNA was extracted with phenol-chloroform, respectively. Next, the degree of enrichment was detected using real-time quantitative PCR.
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