Total cell lysate protein was mixed with 50 μL Protein A/G PLUS-Agarose beads (P2012, Beyotime Biotechnology) at a final concentration of 10 μg/μL. Then, the mixture was centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was incubated with Fyn antibody (30 μg) for 1 hr at RT. Another 30 μL of Protein A/G PLUS-Agarose beads was added to the supernatant and allowed to react at RT for 20 min. The beads were pelleted and washed three times. Fyn kinase activity was assayed with a Universal Tyrosine Kinase Assay Kit according to the manufacturer's protocol (MK410, Takara Bio, Otsu, Japan).
Protein a g plus agarose beads
Manufactured by Beyotime
Sourced in China
Protein A/G plus-agarose beads are a type of affinity chromatography resin used for the purification of antibodies. The beads consist of recombinant Protein A and Protein G covalently coupled to agarose. Protein A and Protein G are bacterial cell wall proteins that have a high affinity for the Fc region of antibodies, allowing for the efficient capture and purification of immunoglobulins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Universal tyrosine kinase assay kit, by Takara Bio (1 mentions)
Sc 805, by Santa Cruz Biotechnology (1 mentions)
B0586, by Agilent Technologies (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti β actin antibody, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Protease inhibitor, by Meilun (1 mentions)
Ripa extraction reagent, by Meilun (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
8 protocols using protein a g plus agarose beads
1
Fyn Kinase Activity Assay
2
Immunoprecipitation and Western Blotting
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Cell lysates were centrifuged (12,000× g) at 4°C for 15 min. Proteins were immunoprecipitated with indicated antibodies and shaken at 4°C overnight. Then, 40 μL precleared Protein A/G Plus-agarose beads (Beyotime Biotechnology, Shanghai, China,) was incubated with immunocomplexes for 3 h at 4°C and centrifuged at 1000× g for 5 min. The supernatant was discarded, and the pellet was washed 5 times with PBS. Next, the precipitates were resuspended with 1× sample buffer and heated at 98°C for 5 min. The samples were centrifuged for 5 s, and the supernatants were collected for Western blotting. Immunoblotting analysis was performed as previously described.
3
HBx Immunoprecipitation and Western Blot Analysis
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At 4 days post-transfection, the cells were collected and lysed on ice in RIPA Lysis Buffer (Beyotime Institute of Biotechnology). The primary antibodies included anti-HBcAg (1:400; B0586; DakoCytomation, Glostrup, Denmark) and anti-HA (1:200, sc-805; Santa Cruz Biotechnology, Inc.). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:5,000; ZB-2010) and the anti-β-actin antibody (1:500; TA-09) were from Beijing Zhongshan Golden Bridge Biotechnology, Co. (Beijing, China). Western blot analysis was performed as previously described (3 (link)), and protein bands were quantitated using the Quantity One Image analysis system. HBx immunoprecipitation was performed prior to western blot analysis. Equal amounts of total protein from cells transfected with pSI-HA-x, pSI-HA-x1-101, pSI-HA-x43-154 and pSI-control were incubated with protein A/G Plus-agarose beads (Beyotime Institute of Biotechnology) and primary anti-HA antibody. Specific operations were performed according to the manufacturer's instructions.
4
ATM Modulates Par6-aPKCι Interaction
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MMH-RT cells were cultured with ATM (0, 10, 50, 100, 200, and 400 μM). After 24 h, cells were washed once with ice-cold PBS and lysed in RIPA lysis buffer with 1 mM PMSF. The protein concentration was determined by Coomassie protein assay (Pierce). In 200 μg of cell proteins, rabbit antibody IgG (1 μg) was dropwise added with protein A/G plus-agarose beads (Beyotime Institute of Biotechnology, Haimen, China) 20 μl, and rotated for 2 h at 4°C to get rid of non-specific combination. The mixture was centrifuged and the supernatant was collected. CO-IP was performed using rabbit anti-aPKCι monoclonal antibody (1 μg) or mouse anti-Par6 monoclonal antibody (1 μg) to bind Par6 protein or aPKCι protein, respectively. The protein-antibody immunoprecipitates were collected by protein A/G plus-agarose beads. The samples were washed with RIPA buffer five times. Following the final wash, the beads were resuspended in 20 μl of SDS loading buffer. Finally, the samples were boiled and centrifuged to pellet the agarose beads. The supernatant was collected for western blot detection using the Par6 antibody (1 : 1000) or aPKCι antibody (1 : 500) and proper second antibody. The intensity of the specific bands was estimated by Image J2X software package. The assays were repeated at least three times.
5
ITCH-ITGB3 Protein Interaction Validation
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Cell lysates were homogenized using RIPA lysis buffer and then incubated with or without anti-ITCH antibody, anti-ITGB3 antibody, or control IgG overnight at 4 °C to confirm the interaction between ITCH and ITGB3. Next, an incubation with protein A/G Plus agarose beads (Beyotime Biotechnology, Shanghai, China, P2019) for 2 h at 4 °C was necessary. After being washed three times in lysis buffer, the immunoprecipitated complexes were analyzed by Western blotting. After being infected with ITCH-OE plasmids, the ectopic ESCs were lysed with RIPA buffer, and the lysates were reacted with anti-ITGB3 or control IgG antibodies, respectively. Finally, the immunoprecipitated complexes were analyzed with Western blotting using anti-ubiquitin.
6
Immunoprecipitation and Immunoblotting Protocol
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Cells were collected after 48 h and lysed with buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), then the lysates were centrifuged at 14,000 g for 10 min at 4 °C. The supernatant was collected and incubated with Anti-Flag antibody overnight at 4 °C, followed by 3 h incubation with protein A/G plus-agarose beads (Beyotime Institute of Biotechnology, Jiangsu, China) at 4 °C. Bead-binding proteins were eluted by boiling in 1x SDS loading buffer for SDS-PAGE and further immunoblot analysis was performed with indicated antibodies. Cell lysates without immunoprecipitation underwent immunoblot analysis with indicated antibodies as the positive control, and proteins precipitated by IgG were used as the negative control.
7
Protein Co-Immunoprecipitation Assay
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Cells were washed with pre-chilled phosphate-buffered saline (PBS) and lysed with RIPA extraction reagent (Meilun, Dalian, China) supplemented with protease inhibitors (Meilun, Dalian, China). Cell lysates were pre-cleared and incubated with indicated antibody overnight at 4 °C, The antibody associated with the protein complex were then incubated with protein A/G PLUS-Agarose beads (beyotime, China) for additional 2 h. The beads were washed with PBS three times and boiled at 100 °C for 10 min to reverse crosslinking before SDS-PAGE immunoblotting analysis.
8
Immunoprecipitation and Immunoblotting of BCL-2
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After washing with PBS, GCs were lysed in 1 mL cold IP lysis buffer (Pierce) containing a complete protease inhibitor cocktail (Roche). Whole-cell lysates were used for immunoprecipitation with anti-BCL-2. We added 2-4 mg of antibody to 1 mL of cell lysate, which was incubated at 4°C overnight. After the addition of Protein A/G Plus Agarose beads (Beyotime Institute of Biotechnology), the incubation was continued for 1 h. Immunoprecipitates were extensively washed with lysis buffer, eluted with SDS loading buffer (SunShineBio, Nanjing, China) by boiling for 5 min and then processed for immunoblotting with the indicated antibodies. The amount of coimmunoprecipitated proteins for each IP reaction was normalized to TUBA1A content in the whole-cell lysates (input).
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