HEK293T cell expressing the proteins of interest in 6-well plates were washed with 1x PBS and lysed with 200 μL HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% NP-40 pH 7.5), supplemented 100X protease inhibitor cocktail (Calbiochem) at 4°C for 1 h and subsequent lysate clearance by centrifugation at 16,000 g at 4°C for 10 min. In case of immunoprecipitation of purified proteins, 2μg of each target protein, (5 μg of GP proteins) mixed with HEPES lysis buffer to a final volume of 20 μL. For cell-free immunoprecipitation purified GP, and commercially available pure MD2 (ab238343, Abcam) and TLR4 (ab233665, Abcam) were incubated with 2 μL anti-MD2 (ab24182, Abcam), anti-TLR4 (ab13867, Abcam) and anti-His (51-9000012, BD-Pharmagen) antibodies at 4°C for 4 h followed by addition of 20 μL protein A/G beads (Abcam) and further 2 h incubation. The beads were washed with HEPES lysis buffer and eluted with 1 M Glycine pH 3 followed by NaOH neutralization.
Protein a g beads
Manufactured by Abcam
Sourced in United States, United Kingdom
Protein A/G beads are agarose beads coated with a combination of Protein A and Protein G, which are bacterial proteins that have a high affinity for the Fc region of antibodies. These beads are commonly used in immunoprecipitation and affinity purification techniques to capture and isolate antibodies from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g bead, by Santa Cruz Biotechnology (3 mentions)
Ab172730, by Abcam (2 mentions)
Anti igg, by Abcam (2 mentions)
Ab24182, by Abcam (1 mentions)
Ab13867, by Abcam (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Ab125938, by Abcam (1 mentions)
Ab275876, by Abcam (1 mentions)
Ip lysate, by Beyotime (1 mentions)
Protein inhibitor, by Solarbio (1 mentions)
Lds sample buffer, by Bio-Rad (1 mentions)
Ab1424, by Abcam (1 mentions)
Ab137452, by Abcam (1 mentions)
Ab5442, by Abcam (1 mentions)
F7425, by Merck Group (1 mentions)
Ab4080, by Abcam (1 mentions)
Ab38113, by Abcam (1 mentions)
Ab9110, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ab205606, by Abcam (1 mentions)
10 protocols using protein a g beads
1
Immunoprecipitation of Protein Complexes
2
ChIP-PCR Assay for β-Catenin Promoter Enrichment
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The ChIP-PCR assay for the enrichment of β-catenin promoter in JMJD2C was performed as previously described (28 (link)) using a ChIP assay kit (cat. no. 17-10086; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, cells were cultured to 90–100% confluence. After washing three times with PBS, cells were cross-linked with 1% formaldehyde (Sigma-Aldrich; Merck KGaA), lysed with 2 ml lysis buffer (50 mM Tris-HCl, pH 8.1/1% SDS/10 mM EDTA protease inhibitors) at 4°C, and sonicated 4×15 times at 4°C. The chromatin fragments were incubated with 3 µg affinity-purified antibodies against JMJD2C (cat. no. ab27532; Abcam) or IgG (cat. no. ab2410; Abcam) at 4°C overnight and precipitated using protein A/G beads (cat. no. sc-2002; Santa Cruz Biotechnology, Inc.) coupled to magna beads. The DNA fragments were extracted using phenol/chloroform and used as templates for PCR. As to PCR, 1 µl from a 50 µl DNA extraction and 38 cycles of amplification were used. The primer sequences for the CTNNB1 promoter was as follows: Forward, 5′-GTAGAGACGGGGTTTCACCA-3′ and reverse, 5′-CCTGGGCAATAAGAGCAAAA-3′. cycle quantification was determined for both immunoprecipitated DNA and known amount of DNA from input sample using the 2−ΔΔCq method (22 (link)). The products were confirmed by 3% agarose gel electrophoresis. Each experiment was performed in triplicate.
3
KRAS and PDE5 Protein Quantification
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Six dishes of Mia PaCa-2 cells were prepared. Renewed the medium with 20 μmol/L compound 36l and 1% DMSO to every 3 dishes respectively and incubated for another 2 h. Harvested and combined the cells, then added 500 μL of IP lysate (Beyotime, containing 1% protease inhibitor) and lysed the cells on ice for 30 min. The prepared Protein A/G beads (Santa Cruz) were added into the cell lysates. Shacked the tubes at 4 °C for 1 h followed by a centrifuge at 4 °C, 12,000 rpm, 15 min. Quantify the supernatant by the BCA method and adjust the total protein concentration to 5 μg/mL. Respectively added KRAS primary antibody (Abcam #ab275876) and IgG (Abcam #ab125938) antibody to 100 μL the above lysates followed by rotating at 4 °C for 1 h. Add the Protein A/G beads 20 μL respectively into the supernatant above, and rotated on the shaker overnight. Centrifuged and collected the precipitate followed by washed it with 500 μL PBS buffer 3 times. Centrifuged and collected the precipitate followed by denaturation in 2 × loading buffer 60 μL at 95 °C for 10 min. Western blotting experiments and gray-scale statistics were conducted to quantify the KRAS and PDEδ protein.
4
Immunoprecipitation of hnRNPU protein
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Cells were collected and washed with PBS. Thereafter, proteins were extracted using RIPA lysis buffer containing protein inhibitor (Solarbio). Afterward, 300 μL extracts were added with 50% Protein A/G beads (Upstate Biotechnology, Lake Placid, NY, USA) for 1 h-incubation at 4°C for pre-clearing. After centrifugation to remove the beads, the supernatant was obtained and incubated overnight with antibody: anti-hnRNPU (1 μg/mL, A300-690A, Thermo Fisher Scientific) or normal rabbit IgG (1:1000, ab172730, Abcam) (negative control) at 4°C, followed by 2 h-incubation with 50% Protein A/G beads at 4°C. Later, the protein-antibody complexes were rinsed with RIPA buffer for 5 min × 5 times, followed by resuspension of protein-antibody complex beads in buffer containing SDS. Immunoprecipitated proteins were boiled at 95–100°C for 3–5 min and the supernatant was collected by centrifugation. The hnRNPU and actin protein levels were then analyzed by WB assay [52 (link),53 (link)].
5
Immunoprecipitation and Western Blot
6
Investigating HSP70-TRAF6 Interaction in Cells
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HEK293T cells were transfected with the plasmids of FLAG-HSP70 and HA-TRAF6 (Public Protein/Plasmid Library, Nanjing, China). After 24 h, cells were harvested with RIPA buffer and incubated with 1 μg anti-HA antibody (ab1424, Abcam) and 20 μL Protein A/G beads (Santa Cruz Biotechnology, USA) overnight at 4 °C. To detect the endogenous interaction of HSP70 and TRAF6, HK-2 cells were lysed and incubated with 3 μg anti-IgG (ab172730, Abcam) or anti-HSP70 (ab5442, Abcam) antibody and 20 μL Protein A/G beads overnight at 4 °C. Proteins were analyzed by Western blotting followed by an anti-HA (1:1000) and anti-FLAG (1:1000; F7425, Sigma) antibody. Endogenous proteins were detected using anti-TRAF6 antibody (1:1000; ab137452, Abcam).
7
Immunoprecipitation and Western Blot Analysis
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The transfected cells were lysed in lysate buffer (mixture of 50 mmol/L Tris ‐ HCl (pH 7.4), 150 mmol/L NaCl, 10% glycerol, 1 mmol/L EDTA, 0.5% NP‐40 and protease inhibitor) and centrifuged to move cell debris. Cleared cell lysate was incubated with 1 μg anti‐HA (ab9110, 1:70, Abcam, Cambridge, UK), myc (ab32072, 1:100, Abcam, Cambridge, UK), USP7 (ab109109, 1:1000, Abcam, Cambridge, UK), KDM6B (ab38113, 1:100, Abcam, Cambridge, UK) or anti‐FLAG antibody (ab205606, 1:1000, Abcam, Cambridge, UK) and 15 μL protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours. After extensive washing, the beads were boiled at 100℃ for 5 minutes. Proteins were resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Temecula, CA, USA), followed by immunoblotting. To detect endogenous protein interactions, cells were lysed in ice‐cold lysis buffer. Cleared cell lysates were incubated with 5 μg anti‐USP7 antibody (ab4080, 1:1000, Abcam, Cambridge, UK) and 20 μL protein A/G beads at 4°C overnight. The anti‐USP7 antibody was used to detect the endogenous KDM6B.
8
Argonaute2-Mediated RNA Immunoprecipitation
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The Magna RIP RNA-Binding Protein Immunoprecipitation kit (cat. no. 17-700; Sigma-Aldrich; Merck KGaA) was used for the RIP assay according to the instructions. Briefly, AMC-HN-3 and Tu177 cells were incubated with Argonaute2 (anti-Ago2; Abcam) or a negative IgG (anti-IgG; Abcam) at 4°C for 2 h. Then, the Ago2 antibody was recovered with the protein A/G beads (cat. no. LSKMAGAG02; Sigma-Aldrich; Merck KGaA). The enrichment of NEAT1, miR-204-5p and SEMA4B was assessed using RT-qPCR.
9
CREB1 Chromatin Immunoprecipitation Assay
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This assay was performed with a ChIP assay kit (Beyotime, Shanghai, China) following the manufacturer's instructions. Briefly, A549 and ATII cells were cross-linked with 1% formaldehyde and sonicated to shear DNA to lengths between 200~1000 base pairs. Cell lysates were incubated at 4°C with protein A/G beads coated with the anti-CREB1 antibody (2 μg, Abcam) or anti-IgG (2 μg, Abcam) for one night. After washing and elution, samples were treated with 5 M NaCl for heating, and then incubated with proteinase K for 1 h. The bound DNA fragments were purified via DNA Extraction Kit (GeneMark, Shanghai, China) and analyzed by real-time PCR.
10
Immunoprecipitation and Western Blot
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After washing twice with pre-cooled PBS, the cells were lysed with pre-cooled RIPA buffer. The lysates were then centrifuged at 14000 g for 15 min at 4°C, and the resulting supernatant was transferred to a new centrifuge tube. The supernatant was incubated overnight at 4°C with 1 μg of primary antibody/20 μl of protein A/G beads (Abcam). The next day, samples were centrifuged at 14000 g for 15 min at 4°C. The supernatant was discarded, and the beads were washed with loading buffer. Subsequently, the beads were boiled at 100°C for 5 min, and Western blotting was performed.
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