For IP experiments, fusion proteins were expressed from pUGW and pFAHW plasmids. One well of a six-well plate of S2 cells at ∼50% confluence was transfected with 0.5 µg DNA using Effectene (QIAGEN) and allowed to recover for 48 h at 25°C. All subsequent steps were performed at 4°C. 50 µl of Protein A–conjugated Dynabeads (Thermo Fisher Scientific) was used per IP and resuspended in 1 ml PBS plus 0.01% Tween 20. 1 µl rabbit anti-GFP antibody (AB290; Abcam) was added and incubated with mixing for 1 h. Cells were harvested and lysed by resuspending in 1 ml RIPA buffer with 1 mM DTT, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 mM PMSF. Lysate was cleared by a 5-min spin in a microfuge at full speed, then incubated with antibody-conjugated beads for 1 h. Beads were washed three times for 5 min each in RIPA buffer. Before the last wash, the beads were moved to a fresh tube. Beads were eluted by boiling in 25 µl of 2× Laemmli buffer for 5 min, and 10-µl samples were then analyzed by SDS-PAGE followed by Western blot.
Protein a conjugated dynabeads
Manufactured by Thermo Fisher Scientific
Protein A–conjugated Dynabeads are superparamagnetic beads coated with Protein A, a bacterial protein that binds strongly to the Fc region of immunoglobulins. These beads are used for the purification and isolation of antibodies and antibody-containing complexes from complex biological samples.
Automatically generated - may contain errors
6 protocols using protein a conjugated dynabeads
1
Immunoprecipitation of Fusion Proteins
2
Chromatin Immunoprecipitation for Oct-1 and Oct-2
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Chromatin samples for ChIP were prepared using a Simple ChIP Enzymatic Chromatin IP Kit (CST) and were precleaned with Protein A-conjugated Dynabeads (Thermo Fisher Scientific) at 4°C for 2 h. Antibodies against Oct-1 (ab15112, Abcam) and Oct-2 (ab179808, Abcam) were bound to Protein A-conjugated Dynabeads, then subjected to immunoprecipitation with the precleaned samples at 4°C overnight. Washing beads and eluting immunoprecipitated DNA fragments were prepared according to the CST manual. qPCR was performed as described above. The qPCR primer sequences were determined based on the Oct-1 or Oct-2 binding sites deposited in the JASPAR and DECODE databases. The qPCR primer sequences for ChIP-PCR are listed in Table 3 . Data were normalized using the Ct values of input samples.
3
Ovarian Protein Immunoprecipitation Protocol
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Dissected ovaries (> 50 pairs) were homogenized in 1.5ml tubes and proteins extracted using 300mM KCl buffer D. Equal amounts of ovarian extract were incubated with antibodies overnight, followed by incubation with Protein A-conjugated Dynabeads (Invitrogen) for 3 hours. Beads were then washed with 0.05% T/PBS, and immunoprecipitates eluted by 2X loading buffer.
4
Ovarian Protein Immunoprecipitation Protocol
5
Immunoprecipitation and Kinase Assay for Polo Proteins
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For immunoprecipitation of Polo-GFP (WT or mutants), extracts were prepared as above and lysates were incubated with anti-GFP antibodies (#A6455, Invitrogen) for 1 h at 4 °C and then incubated with 20 μL of Protein A-conjugated Dynabeads (Life Technologies) for 45 min at 4 °C, before being washed in lysis buffer as above.
For kinase assays, reactions were performed in kinase buffer (20 mM K-HEPES pH 7.5, 2 mM MgCl2, 1 mM DTT) with 0.5 μM ATP,32P-γ-ATP, and 1 μg casein at 30 °C for 15 min with agitation. For Polo inhibition in the kinase assay, BI 2536 was added at 300 nM. Reactions were stopped with the addition of the Laemmli buffer and heating at 95 °C for 2 min. Samples were separated by SDS–PAGE and transferred onto nitrocellulose membranes for autoradiography and western blotting with anti-GFP antibodies (#1218, Abcam).
For kinase assays, reactions were performed in kinase buffer (20 mM K-HEPES pH 7.5, 2 mM MgCl2, 1 mM DTT) with 0.5 μM ATP,32P-γ-ATP, and 1 μg casein at 30 °C for 15 min with agitation. For Polo inhibition in the kinase assay, BI 2536 was added at 300 nM. Reactions were stopped with the addition of the Laemmli buffer and heating at 95 °C for 2 min. Samples were separated by SDS–PAGE and transferred onto nitrocellulose membranes for autoradiography and western blotting with anti-GFP antibodies (#1218, Abcam).
6
Immunoprecipitation of Akt1 from Synaptosomes
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Immunoprecipitation of Akt1 was carried out using Protein A-conjugated Dynabeads (Life Technologies) according to the manufacturer's instructions. In brief, Protein A-conjugated Dynabeads were incubated with anti-Akt1 antibody in PBS (pH 7.4) containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.1% (v/v) Igepal CA360 for 3 h at room temperature. The antibody-linked Dynabeads were washed with PBS and incubated overnight at 4°C with synaptosomes in PBS containing 1% (v/v) Triton X-100 and protease and phosphatase inhibitors. The beads were washed twice with PBS containing 0.5% (v/v) Igepal CA360 at 4°C and eluted using Laemmeli's SDS loading buffer and subjected to SDS PAGE.
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