[35S]Met-labeled preproteins synthesized in RRL (Promega, United States) were incubated with rotation for 1 h at RT in the presence of preimmune sera (GenScript, United States), anti-14-3-3 (a gift from Prof. Carol MacKintosh, University of Dundee) or anti-Hsp70 antibodies (Catalog no.: AS08 371; Agrisera, Sweden) in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 0.2% Triton X-100, pH 7.5) (Dessi et al., 2003 (link)). The antibodies were prepared against KLH-conjugated synthetic peptide conserved in the five cytosolic Hsp70 proteins of Arabidopsis thaliana (Sung et al., 2001 (link)). The bound proteins were pulled down using Protein-A Sepharose beads (Thermo Scientific, United States) and washed by reaction buffer containing 250 mM NaCl four times. After the supernatant was discarded, the Protein-A Sepharose beads (Invitrogen, United States) was resuspended in Lammeli sample buffer and denatured at 95°C for 5 min. The immunoprecipitated proteins were analyzed in SDS-PAGE gel and detected by autoradiography.
Protein a sepharose beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protein A-Sepharose beads are a type of chromatography resin used for the purification of antibodies. The beads are composed of Sepharose, a cross-linked agarose, with covalently attached Protein A, a bacterial protein that binds to the Fc region of immunoglobulins. These beads are commonly used in affinity chromatography to selectively capture and purify antibodies from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Gelcode blue, by Thermo Fisher Scientific (3 mentions)
Seeblue plus 2 marker, by Thermo Fisher Scientific (3 mentions)
Anti h3k4trimethyl antibody, by Abcam (3 mentions)
Isotype control rabbit polyclonal igg, by Merck Group (3 mentions)
Sample buffer, by Bio-Rad (2 mentions)
Protein a sepharose beads, by Merck Group (2 mentions)
Magnetic bead, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Avantor (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Anti flag agarose beads, by Merck Group (2 mentions)
Cw0098m, by CWBIO (2 mentions)
Protease inhibitors cocktail, by Merck Group (2 mentions)
Pwo polymerase, by Roche (2 mentions)
Nuclease treated rabbit reticulocyte lysate, by Promega (2 mentions)
T7 rna polymerase, by Promega (2 mentions)
Rnasin, by Promega (2 mentions)
35s methionine, by PerkinElmer (2 mentions)
Np 40, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
85 protocols using protein a sepharose beads
1
Immunoprecipitation of Radiolabeled Preproteins
2
Kinase Activity Profiling of ALK Variants
3
Protein Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 8.0/150 mM NaCl/0.5% NP-40/10% glycerol/Protease Inhibitor Cocktail (Roche)/50 U/mL of benzonase nuclease (Sigma)) for 1 h. Lysate was spun 13,000 × g for 30 min at 4 °C and pre-cleared with protein A-Sepharose beads (Life Technologies) for 1 h. Then, the pre-cleared lysates were incubated with rabbit IgG, SON antibody or RBFOX2 antibody and protein A-Sepharose beads at 4 °C overnight. Beads were washed four times with the lysis buffer and eluted by boiling in SDS buffer and subjected to Western blot analyses.
4
Immunoprecipitation of Protein Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were centrifuged and the supernatant was preincubated with 20 μg of protein A-Sepharose beads (Thermo Fisher Scientific) with vibrating for 2 h at 4°C. The samples were then centrifuged again and transferred to a new 2-ml EP tube. The samples were incubated with the primary antibodies for 1.5 h and then incubated with protein A-Sepharose beads to capture the immunocomplex. Next, the beads were washed three times, dissolved in electrophoresis sample loading buffer, and incubated for 15 min at 95°C.
5
Rituximab BB-37 Fc Binding Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 28
Duplicate samples of Rituximab or Rituximab BB-37 (100 ul, 50 ug/ml in PBS) were incubated with 12.5 ul protein A sepharose beads (Thermo Fisher 22810) with rotating overnight. Beads were pelleted by centrifugation, supernatant was removed, and residual liquid was removed from the beads using a fine pipette tip. Non-reducing Laemmli sample buffer (100 ul) was added to the beads. Beads and supernatants were heated to 90° C. for 5 minutes and equal fractions were analyzed by SDS-PAGE (4-12% NUPAGE gel, MOPS buffer) followed by staining with Coomassie (GelCode™ Blue, ThermoFisher). Molecular weight standard is SeeBlue® Plus 2 marker (ThermoFisher LC5925). As seen in
6
FcRN Binding Assay for Rituximab BB-37
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 28
Duplicate samples of Rituximab or Rituximab BB-37 (100 ul, 50 ug/ml in PBS) were incubated with 12.5 ul protein A sepharose beads (Thermo Fisher 22810) with rotating overnight. Beads were pelleted by centrifugation, supernatant was removed, and residual liquid was removed from the beads using a fine pipette tip. Non-reducing Laemmli sample buffer (100 ul) was added to the beads. Beads and supernatants were heated to 90° C. for 5 minutes and equal fractions were analyzed by SDS-PAGE (4-12% NUPAGE gel, MOPS buffer) followed by staining with Coomassie (GelCode™ Blue, ThermoFisher). Molecular weight standard is SeeBlue® Plus 2 marker (ThermoFisher LC5925). As seen in
7
Chromatin Immunoprecipitation for Epigenetic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assay was performed as described previously (Ishii et al., 2009 (link)). Briefly, cells fixed in paraformaldehyde were lysed and sonicated to generate 100–300-bp fragments. Immunoprecipitated samples were incubated in anti-H3K27trimethyl antibody (Active Motif; 39155) or isotype control (Millipore; rabbit polyclonal IgG) in parallel samples overnight followed by addition of protein A Sepharose beads (Thermo Fisher). Bound DNA was eluted and purified using phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Primers were designed using the Ensembl genome browser to search the IL-1β, IL-12, and IL-23 promoter for NF-κB within the promoter region, and then NCBI Primer-BLAST was used to design primers that flank this site. Data are representative of two or three independent experiments. Primer sequences are available in Table S3 .
8
ChIP Assay for Protein-DNA Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin Immunoprecipitation (ChIP) assays were conducted according to standard protocols published by ABcam. Following treatments, cells were incubated with formaldehyde at a final concentration of 0.75% for 10 minutes followed by glycine at a final concentration of 125 mM for an additional 5 minutes. Cells were then washed two times with Phosphate Buffered Saline (PBS) and lysed in FA lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS, protease inhibitors). Resulting cell lysates were sonicated to fragment DNA, spun down, and incubated with antibody conjugated protein A sepharose beads (ThermoFisher cat# 101041) overnight with gentile agitation. Following incubation, beads were pelleted, washed three times with FA lysis buffer, and DNA was eluted with elution buffer (1% SDS, 100 mM NaHCO3). Resulting DNA fragments were further purified with a DNA purification kit (Clontech cat# 740609.250) prior to qPCR analysis. Antibodies used were anti-APP C-terminus (Sigma #A8717); anti-FOXO1 (Abcam #39670). Primer sequences listed in Table S1 .
9
GATA3 Chromatin Immunoprecipitation and qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP and qChIP analyses were performed using an EZ-ChIP kit (EMD Millipore) according to the manufacturer's instructions. Briefly, Hep3B and MHCC97-H cells were cultured to 80–100% confluence and the chromatin was cross-linked by 1% formaldehyde at 37°C for 10 min. Subsequently, cross-linked chromatin was sonicated (20 kHz; amplitude, 40%; 30 cycles, 1 sec on and 1 sec off) at 4°C to generate 200–1,000 bp fragments. Next, 4 µg of anti-GATA3 (cat. no. ab199428; Abcam) or anti-IgG antibody (cat. no. ab171870; Abcam) were used to immunoprecipitate chromatin fragments at 4°C overnight. IgG antibody was used as the control. The protein-DNA complexes were incubated with protein A Sepharose beads (Thermo Fisher Scientific, Inc.), and eluted in 1% SDS/0.1 M NaHCO3. The protein-DNA cross-link was reversed by heating at 65°C for 6 h. The DNA was purified using QIAEX II Gel Extraction kit (Qiagen GmbH) according to manufacturer's instructions. After purifying the antibody-interact DNA, RT-qPCR was conducted to analyze the precipitated chromatin DNA, as aforementioned. The primer sequences were as follows: Slug forward, 5′-TCCGGTGGTTCCAAATGACA-3′; and reverse, 5′-TCCGGTGGTTCCAAATGACA-3′. The qPCR conditions were as follows: 5 min at 98°C, denaturation at 98°C for 30 sec, annealing at 56°C for 30 sec and extension at 72°C for 20 sec, performed for 32 cycles.
10
Immunoprecipitation of Axin and IFT Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates from 120 wing imaginal discs of tub > AxinGFP, tub > IFT122GFP, tub > IFT140GFP (1 mg of total protein) were precleared by incubating with protein A-sepharose beads (Thermo Scientific) for 1 h at 4 °C followed by centrifugation. A-sepharose beads were immunoprecipitated with specific antibodies at 4 °C for 1 h. Polyclonal anti-GFP antibody (Roche) was used. Immunoprecipitates were captured by protein A-sepharose at 4 °C in IP buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 100 mM NaCl, 0.05% Triton X100, 1 mM EDTA, 5 mM DTT, 10 mM NaVO4, 10% glycerol and protease inhibitor cocktail). Immunoprecipitates were resuspended in SDS sample buffer, boiled for 5 min, separated by SDS-PAGE, and transferred to nitrocellulose for immunoblotting. Protein was detected by enhanced chemiluminescence (Millipore).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!