Co-immunoprecipitation to assess for HMGB1/IL-1β complexes in plasma was performed using the Catch and Release® kit from Millipore according to the manufacturer’s instructions. Briefly, 500μg of sample was incubated in the spin column with anti-HMGB1 antibody and the included antibody capture affinity ligand for 30 minutes. The flow through was collected by centrifugation of the spin column. The column underwent multiple washes followed by elution. The eluate was assessed by Western Blot for HMGB1 and IL-1β.
Catch and release kit
Manufactured by Merck Group
Sourced in United States
The Catch and Release kit is a laboratory equipment designed for the collection and release of samples. It provides a controlled and contained environment for handling various types of samples. The core function of the kit is to facilitate the safe and efficient transfer of samples from one location to another.
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Lab products found in correlation
Anti nt 3, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti bdnf, by Santa Cruz Biotechnology (1 mentions)
Anti β actin ab, by Merck Group (1 mentions)
Genesnap, by Syngene (1 mentions)
Mouse anti parp1, by Santa Cruz Biotechnology (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Anti p75ntr, by Santa Cruz Biotechnology (1 mentions)
Anti phospho trka y490, by Cell Signaling Technology (1 mentions)
Anti ngf, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti hmgb1, by Abcam (1 mentions)
Antibodies against acetyl lysine, by Cell Signaling Technology (1 mentions)
8 protocols using catch and release kit
1
Detecting HMGB1/IL-1β Complexes in Plasma
2
Neurotrophin Signaling Pathway Analysis
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Western blotting was performed as described previously (Bellanger et al, 2011 (link)). Rabbit anti-BDNF, anti-NGF, anti-NT3, anti-p75NTR, goat anti-sortilin, and mouse anti-PARP-1 antibodies were purchased from Santa Cruz Biotechnology. Mouse anti-TrkB, anti-TrkA, and anti-TrkC antibodies were purchased from R&D Systems. Anti-phospho TrkB (pY817) and anti-phospho-TrkC (Y820) were purchased from Abcam (Epitomics, Burlingame, CA, USA) and anti-phospho-TrkA (Y490) from Cell Signalling. Protein-loading control was performed with anti-βactin Ab (Sigma). Visualisation of immunocomplexes was accomplished using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Darmstadt, Germany). Western blots were scanned using a bioimaging system (Genesnap; Syngene Europe, Cambridge, UK).
Co-immunoprecipitations were performed in accordance with the manufacturer's instructions (Catch and Release kit, Millipore) with 500 μg of proteins and anti-p75NTR for 1 h at room temperature. Finally immunoprecipitates were subjected to SDS–polyacrylamide gel electrophoresis, before analysis by western blotting (with anti-Trk, anti-sortilin and anti-p75NTR).
Co-immunoprecipitations were performed in accordance with the manufacturer's instructions (Catch and Release kit, Millipore) with 500 μg of proteins and anti-p75NTR for 1 h at room temperature. Finally immunoprecipitates were subjected to SDS–polyacrylamide gel electrophoresis, before analysis by western blotting (with anti-Trk, anti-sortilin and anti-p75NTR).
3
HMGB1/IL-1β Protein Complexation Assay
4
Detecting Acetylated HMGB1 in Culture Medium
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Immunoprecipitation was performed to detect the status of acetylated HMGB1 in culture medium using a catch and release kit (Millipore, USA) according to the manufacturer’s instructions. Briefly, a total 250 µl of culture medium from each sample was incubated with 5 µg of antibodies against acetyl-lysine (Cell Signaling, USA) and 10 µl affinity ligand for 1 hr at room temperature. Proteins were eluted in its native form and subjected to Western blot analysis. The blots were probed with rabbit anti-HMGB1 (1∶500, Abcam).
5
Protein Extraction and Immunoprecipitation
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Total proteins were extracted in CHAPS buffer (10 mM Hepes, 150 mM NaCl, 1% CHAPS, pH 7.4 and EDTA-free protease inhibitors cocktail) as previously described and were immunoprecipitated with the Catch and Release kit, as described by the manufacturer (Millipore).
6
Myocilin Ubiquitination in Human TM Cells
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Lysates from human TM cells untreated or treated with 1 µM LCT for 16 h were immunoblotted using polyclonal anti-myocilin or monoclonal anti-ubiquitin (1∶2000, Biomol, Enzo Life Sciences). Lysates were also immunoprecipitated with polyclonal anti-N-terminal-myocilin (a generous gift from Dr. W. Daniel Stamer, Duke University) or rabbit normal IgG (negative control) using the Catch and Release kit (Millipore, Billerica, MA). The proteins pulled down were subjected to SDS-PAGE under reducing conditions. The ubiquitinated proteins were detected with mouse anti-ubiquitin antibody.
7
Co-immunoprecipitation of Tβ4 Protein
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Co-immunoprecipitation was performed according to the instructions of the Millipore Catch and Release kit. Briefly, samples containing 1–3 mg total protein were precleared with Protein A/G Sepharose for 30 min. The cleared lysates were incubated with 4 mg anti-Tβ4 at 4 °C for at least 2 h, and up to overnight [37 (link)].
8
Irradiation-Induced p53 Phosphorylation Analysis
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For protein expression analysis, western immunoblotting was performed at different time points after irradiation, as detailed earlier50 (link). For p53 immunoprecipitation cells were irradiated with sub-lethal and lethal doses and harvested 24 h post-irradiation. Cells were lysed using native lysis buffer (150 mM NaCl, 10 mM HEPES [pH 7.4], 1% CHAPS, 10% Protease inhibitor cocktail) and 250 μg of whole cell lysate was used for immunoprecipitation using Catch and Release kit (Millipore, USA) as per manufacturer’s protocol. Eluted proteins were separated on 15% PAGE and transferred onto PVDF membrane. Detection of phosphorylated Sfp53 was performed using phosphor- Serine, Threonine specific antibodies and Enhanced Chemiluminescence (ECL) reagent (Pierce, USA). The position of phospho-Sfp53 was marked by p53 specific antibody on the same membrane.
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