DLBCL (9 × 106 cells) were lysed in 2 ml of Triton X-100 buffer (50 mM Tris-HCL [pH 7.4], 150 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 10% Glycerol, 1% Triton X-100 and 1x protease inhibitor cocktail tablets [Roche]) on ice for 30 min and then centrifuged at 18,000 × g for 20 min at 4°C. 400 μg proteins was pre-cleared using 30 μl of Pierce Protein A Magnetic Beads (Thermo Scientific) for 2 hours at 4°C then incubated overnight at 4°C with MCL-1 antibody (Protein Tech, 16225), BCL-2 antibody (Epitomics, 1017–1) or Rabbit IgG. 20 μl of Magnetic beads were added and incubated for 2 hours. Immunoprecipitates were then washed 5 times with 500 μl of Triton X-100 buffer and boiled in NuPAGE LDS Sample Buffer (Thermo Scientific) supplemented with 1 mM DTT and proteins were separated on NuPAGE 10% Bis-Tris polyacrylamide gels.
Pierce protein a magnetic beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pierce Protein A Magnetic Beads are a type of magnetic bead-based affinity chromatography resin designed for the purification of antibodies. The beads are coated with recombinant Protein A, which binds to the Fc region of immunoglobulins, allowing for the capture and isolation of antibodies from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor, by Roche (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Protein a magnetic beads, by New England Biolabs (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Dykddddk tag monoclonal antibody fg4r, by Thermo Fisher Scientific (1 mentions)
Control igg antibody, by Cell Signaling Technology (1 mentions)
Pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Dynamag 2 magnet, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Phosstop, by Merck Group (1 mentions)
Mini edta free, by Roche (1 mentions)
Easypack, by Roche (1 mentions)
Phosstop easypack, by Roche (1 mentions)
Complete tablet, by Roche (1 mentions)
Pi 3 p, by Echelon Biosciences (1 mentions)
13 protocols using pierce protein a magnetic beads
1
Immunoprecipitation of BCL-2 Family Proteins
2
Purification of Recombinant IgG
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IgG was induced in SHuffle2, cells were harvested and the cell pellet was resuspended into 1 mL lysis buffer (1XPBS with 5% glycerol and 1 mM EDTA) to OD 50. The 1 mL cell suspension was sonicated with mini tipped sonicator for 1 min (4 s on and 2 s off) and repeated three times. The sonicated cell suspension was spun at 13,000 rpm for 15 min. The supernatant containing the soluble fractionation was separated from the insoluble pellet and subjected to purification. IgG was purified from the soluble fraction by affinity using protein A magnetic beads (New England Biolabs, cat. No. S1425) following the “Antibody Purification” protocol as recommended in the Pierce™ protein A magnetic beads manual (ThermoFisher, MAN0011856), using Thermo Scientific KingFisher Flex instrument (ThermoFisher, 5,400,640). The low pH of the eluate was neutralized by adding 5 μL of Neutralization Buffer (20% Tris base pH 9.5) and purified fraction was resolved by SDS-PAGE under non-reducing and reducing conditions. Proteins bands were visualized by staining with SimplyBlue™ SafeStain (ThermoFisher Scientific, Invitrogen™, cat. No. LC6065). And yields of purified IgG was obtained by BCA assay (ThermoFisher, cat 23,225).
3
Immunoprecipitation of H2B Variants and SPT16-GFP
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Cell extracts from H2B WT and H2B R95A were extracted in yeast suspension buffer (YSB) supplemented with protease inhibitors. Protein extracts were incubated with anti-GFP antibodies (Roche Antibodies) at 4 °C overnight and then the immune complex was conjugated to pierce protein A magnetic beads (ThermoFisher Scientific) for 2 h at room temperature. After IP, the beads were washed 4 times thoroughly with TRIS buffered saline containing 0.01% Tween 20 and protease inhibitors. Immunoprecipitated proteins were eluted using 2 × SDS loading buffer and then boiled at 95 °C for 5 min. Denatured proteins were subsequently separated on SDS PAGE gels and immunoblotted against anti-FLAG antibodies (Sigma-Aldrich). For SPT16-GFP tagged strains, cell extracts were incubated with Chromotek-GFP trap magnetic agarose beads (Proteintech) for 2 h at 4 °C. after IP, the beads were washed three times with the wash buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.05% Nonidet™ P40 Substitute, 0.5 mM EDTA) supplemented with protease inhibitors. Proteins were eluted in 2 × SDS loading buffer and then boiled at 95 °C for 5 min. Samples were loaded on SDS PAGE gels and blotted against anti-FLAG monoclonal antibody (DYKDDDDK tag Monoclonal antibody (FG4R), MA1-91878, Invitrogen, USA).
4
Immunoprecipitation and Western Blot Analysis
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BMDMs were treated with Dex or 911 as described above and washed with PBS containing pStop (Roche) and Complete Protease inhibitor (Roche). Cells were lysed using RIPA buffer and protein content was measured using BCA. Immunoprecipitation was performed with 300 μg of proteins using Pierce Protein A Magnetic Beads (Thermo Fisher Scientific). Protein lysates were precleared by incubation with protein A beads followed by incubation with 1:100 anti‐pSerine AB (Novus NB100‐1953) or 3 μg of IgG control antibodies (Cell Signaling Technology) overnight on a rotator at 4°C. Beads were washed with PBS and resuspended in 1× SDS buffer, which was then boiled for 5 min. Leftover beads were removed using a magnet. Western blot was then performed as described above. IgG band intensity was removed from calculations.
5
Brassinosteroid-Regulated Chromatin Immunoprecipitation
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For ChIP assays, 5 day-old Arabidopsis seedlings (pBZR1::BZR1-CFP and negative control 35S::YFP lines) grown in the dark were treated with 100 nM BL (24-Epibrassinolide) for 1 h and cross-linked for 20 min in 1% formaldehyde under vacuum. The chromatin complex was isolated and resuspended in lysis buffer (50 mM HEPES pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1X protease inhibitor) followed by sonication to reduce the average DNA fragment size to a range of 200-500 bp. The sonicated chromatin complex was immunoprecipitated using an anti-YFP antibody (custom made) bound to Pierce protein A magnetic beads (Thermo Scientific, Prod #88846, Lot#NK180758). The beads were washed with low-salt buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100), high-salt buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 0.5% Triton X-100), LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25 M LiCl, 0.5% NP-40, 0.5% deoxycholate) and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and eluted with elution buffer (1%SDS, 0.1 M NaHCO3). After de-crosslinking and DNA recovery, DNA was purified using a PCR purification kit (Thermo Scientific) and analyzed by qPCR. The enrichment of DNA was calculated as the ratio between BZR1-CFP and 35S::YFP samples, normalized to that of the CNX5. Primers for qPCR are listed in Table S1 .
6
Rubicon Immunoprecipitation and Analysis
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For exogenous Rubicon immunoprecipitation: 293T cells were transfected with empty vector, pcDNA6-GFP-FLAG-Rubicon WT, and pcDNA6-GFP-FLAG-Rubicon S92A. MMTV-neu cells were engineered to stably express pBabepuro-Rubicon WT-Flag and pBabepuro-Rubicon S92A-Flag. Flag-Rubicon WT and S92A proteins were immunoprecipitated from cell lysates using anti-Flag-M2 Magnetic Beads (Sigma-Aldrich #M8823) at 4°C with rotation overnight. For this analysis, lysis buffer as described above also contained 0.1% SDS. Protein bound beads were washed 3 times in lysis buffer and resuspended in 2X-protein sample buffer before being used for immunoblotting analysis as described above. For endogenous Rubicon immunoprecipitation: MMTV-neu Hunk WT and Hunk KO cells and JIMT-1 cells were immunoprecipitated from cell lysates using Pierce Protein A Magnetic Beads (ThermoFisher Scientific) at 4°C with rotation overnight. For this analysis, lysis buffer was used as described above. Protein bound beads were washed 3 times in lysis buffer and resuspended in 2X-protein sample buffer before being used for immunoblotting analysis as described above.
7
Co-Immunoprecipitation with Protein A Beads
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For co-immunoprecipitation (co-IP), Pierce™ Protein A Magnetic Beads (Thermo Fisher, #88845) were used and the experiments were performed according to the manufacturer’s protocol with slight modifications. Briefly, pre-cleared whole cell lysates were incubated overnight with primary antibody at 4 °C with constant mixing. Then, pre-washed protein A beads were added to the antigen-antibody complex and mixed at room temperature for an hour. The antigen-antibody complex bound beads were separated from the mix using the DynaMag™-2 Magnet (Thermo Fisher, #12321D) and were washed three times with Tris-buffered Saline (TBS) containing 0.01% Tween-20 (Sigma, #822184). The beads were washed once with sterile water. Then bound proteins were eluted by mixing beads with 2× Laemmli SDS sample buffer and were denaturized at 100 °C for 10 min in a heat block. SDS-PAGE and western blotting experiments were then performed.
8
Immunoprecipitation of Kib-GFP
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pMT-Kib-GFP and pAHW-Par-1 or pAHW-Par-1KD (gift from Bingwei Lu, Stanford University) were transfected and expression was induced as described above. Immunoprecipitation (IP) was performed 3 d after transfection. To induce expression of pMT-Kib-GFP, 700 mM CuSO4 was added to the wells 24 h before cell lysis (2 d after transfection).
Cells were harvested and lysed on ice in buffer containing 25 mM HEPES, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.5 mM ethylene glycol-bis(b-aminoethyl ether)-N, N, N0, N0-tetraacetic acid, 0.9 M glycerol, 0.1% Triton X-100, 0.5 mM Dithiothreitol, and Complete protease inhibitor (Roche) and PhosSTOP (Sigma Aldrich) phosphatase inhibitor cocktails at one tablet/10 ml concentration each. Cell lysates were then incubated with anti-GFP antibody (guinea pig, seeTable 1 ) for 30 min. Antibody-bound Kib-GFP was pulled down using Pierce Protein A magnetic beads (Thermo Fisher Scientific) for 1.5 h. A control immunoprecipitated sample was treated with λ-phosphatase. Samples were run on 8% polyacrylamide gel, with 118:1 acrylamide/bisacrylamide (Scheid et al., 1999 (link)), to better resolve phosphorylated Kib species. Kib was detected by Western Blot using anti-GFP antibody (rabbit, see Table 1 ).
Cells were harvested and lysed on ice in buffer containing 25 mM HEPES, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.5 mM ethylene glycol-bis(b-aminoethyl ether)-N, N, N0, N0-tetraacetic acid, 0.9 M glycerol, 0.1% Triton X-100, 0.5 mM Dithiothreitol, and Complete protease inhibitor (Roche) and PhosSTOP (Sigma Aldrich) phosphatase inhibitor cocktails at one tablet/10 ml concentration each. Cell lysates were then incubated with anti-GFP antibody (guinea pig, see
9
Immunoprecipitation and Western Blot Protocol
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For CoIP, protein was extracted by Pierce IP Lysis Buffer (87787, Thermo Fisher Scientific) with cOmplete Tablets, Mini EDTA-free, EASYpack (04693159001, Roche, Basel, Switzerland), and PhosSTOP EASYpack (04906837001, Roche). Briefly, the lysates were incubated with certain antibodies at 4°C overnight. Then, lysates were incubated with Pierce Protein A Magnetic Beads (88846, Thermo Fisher Scientific) for an hour at room temperature. After being washed by lysis buffer, beads were heated with SDS loading and analyzed by WB. Antibodies used in this study were listed in table S6.
10
Lipid Reagent Procurement and Handling
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Lipids were purchased from Avanti Polar Lipids (Alabaster, AL) with the exceptions of PI3P (p hosphatidylinositol 3-phosphate diC16) from Echelon Biosciences (Salt Lake City, UT) and ergosterol from Fluka (St. Louis, MO). Octylglucoside (n-octyl-ß-d -glucopyranoside) was from Anatrace (Maumee, OH), Cy5-labeled streptavidin from LGC Clinical Diagnostics (Milford, MA), biotin-conjugated R-phycoerythrin from Life Technologies Corporation (Eugene, OR), and Histodenz from Sigma-Aldrich (St. Louis, MO). Dialysis tubing was from Repligen Corporation (Waltham, MA) and Bio-Beads SM-2 Resin from Bio-Rad Laboratories (Hercules, CA). Underivatized streptavidin and Pierce Protein-A Magnetic Beads were from Thermo Scientific (Waltham, MA).
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