To deplete Wif1 from OPC conditioned medium, APCfl/fl control or Olig2cre:APC fl/fl OPC conditioned medium were incubated with Wif1 antibody (1:100, rabbit, ab155101, Abcam) at 4℃ overnight followed by pull down with agarose beads (sc-2003, Santa Cruz). BSA was used as the non-antibody control. The purified Wif1-depleted conditioned media were then used for endothelial cell treatment. To clarify the concentrations of Wif1 protein in the conditioned media before and after Wif1 depletion, four groups of conditioned media (WT-CM+BSA, Olig2cre:APC fl/fl-CM+BSA, WT-CM+Ab and Olig2cre:APC fl/fl-CM+Ab) were examined using the Wif1 specific ELISA kit (mouse WIF1 PicoKine Elisa Kit, EK1523, Boster) according to the manufacturer’s instructions. The OD values were determined by measuring the absorbance at 450nm using the microplate reader (Bio-RAD, Model 680). Independent experiments were performed in triplicate.
Agarose beads
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Agarose beads are porous, cross-linked polysaccharide particles used as a support matrix in various chromatographic techniques. They provide a high surface area and are inert, making them suitable for the separation, purification, and immobilization of biomolecules such as proteins, enzymes, and nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (3 mentions)
Ab155101, by Abcam (2 mentions)
Mouse wif1 picokine elisa kit, by Boster Bio (2 mentions)
Model 680, by Bio-Rad (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Anti mouse igg antibody, by Santa Cruz Biotechnology (2 mentions)
Agarose beads, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose transfer membranes, by Santa Cruz Biotechnology (2 mentions)
Ecl kit, by Santa Cruz Biotechnology (2 mentions)
Anti hmgb1 antibody, by Santa Cruz Biotechnology (2 mentions)
Quantity one imaging analysis, by Bio-Rad (2 mentions)
Anti fip200 antibody, by Cell Signaling Technology (2 mentions)
Anti acetylation on epsilon amine groups of lysine residues antibody, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Bio-Rad (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Solarbio (1 mentions)
51 protocols using agarose beads
1
Wif1 Depletion from OPC Conditioned Media
2
HLA Class I Immunopurification and Peptide Elution
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monoallelic KRAS-TMC K562 and 721.221 cell lines were expanded to 1–2 × 108 total cells and HLA class I immunopurification (IP) was performed31 (link) using MHC class I (W6/32) antibody non-covalently linked to agarose beads (Santa Cruz Biotechnology, Dallas, TX). Peptides were eluted from HLA class I molecules using 0.1% trifluoroacetic acid (TFA). Immunoprecipitation eluent was passed through a 10,000 Da Amicon molecular weight cut off filter (Merck Millipore) via centrifugation at 10,000 × g for 10 min. Filtered eluent was dried and resuspended in 100 μl of 0.1% TFA. Stage tip C18 columns (Harvard Apparatus) were equilibrated with 200 μl of acetonitrile and 200 μl of 0.1% TFA, and samples were loaded onto columns for desalting. Samples were then washed with three rounds of 200 μl of 0.1% TFA then eluted in 70% acetonitrile in 0.1% formic acid (FA) and dried.
3
Etoposide-Induced WRN Acetylation Assay
4
Affinity Purification of LAIR-1 Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SKOV3 cells infected with LAIR-1-lentivirus (OE) or control-lentivirus (NC) were lysed in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors. The total cell extracts were centrifuged at 16,000 ×g for 15 min at 4°C, and the supernatant was incubated with anti-Flag antibody overnight at 4°C, then incubated with agarose beads (Santa Cruz, #sc-2003) at 4°C for 4 h, mouse IgG as a non-specific control. The resulting agarose beads were washed three times with 1×PBS and the Flag-associated and non-specific IgG-associated protein complexes were loaded into a 10% SDS-PAGE gel for electrophoresis. Proteins were stained with a coomassie brilliant blue staining solution and then destained with the destaining solution (Solarbio, #P1305).
5
Immunoprecipitation and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and lysed with RIPA buffer (Solarbio) at 4°C. The cell lysates were mixed with indicated antibody at 4°C overnight. The next day, protein A/G plus agarose beads (Santa Cruz Biotechnology) were added; the mixture was then rotated for 2 h at 4°C. Next, the beads were washed four times with RIPA buffer and boiled for 7 min in loading buffer. The eluted protein was subjected to Western blotting.
6
Immunoprecipitation of TRAF6 and USP4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoprecipitation (IP), HEK293T cells that were co-transfected with Flag-TRAF6 and HA-USP4 for 48 h were harvested and lysed on ice with a cell lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 0.5 mM DTT, 1 mM Na3VO4, and 25 mM β-glycerol-phosphate, together with 1 mM PMSF, a complete protease inhibitor cocktail (Roche, Basel, Switzerland), and a phosphatase inhibitor cocktail (Roche). Whole cell lysates were collected by centrifugation for 20 min at 12,000 × g and were incubated with a HA antibody or Flag antibody for 1 h at 4 °C. This was followed by an overnight incubation with protein A/G plus agarose beads (Santa Cruz) at 4 °C. After which the beads were collected via centrifugation for 1 min at 3000 × g and washed six-times with the lysis buffer. The immunoprecipitants were eluted from the beads and subjected to western blot analysis; 1–2% whole cell lysates (Input) served as a control for the IP.
7
Immunoprecipitation of Protein Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells cultured in 10-cm plates of 95% confluency were lysed in co-immunoprecipitation lysis buffer (Beyotime), briefly sonicated and centrifuged for clarification. Proteins were quantified using a BCA assay kit, then incubated with either agarose beads (Santa Cruz Biotechnology) and the intended antibodies including anti-APE1 antibody (Santa Cruz Biotechnology), anti-YBX1 antibody (Cell Signaling Technology), anti-PLK1 antibody (Proteintech), and anti-PPP1R12A antibody (Proteintech) or IgG (Proteintech) or with Anti-flag M2 beads (Sigma-Aldrich) at 4 °C overnight. Beads were washed four times with co-immunoprecipitation buffer with rotation at 4 °C for 5 min each time, boiled with 5× SDS loading buffer (Beyotime), clarified by centrifugation and loaded onto an SDS-PAGE gel for WB analysis as described above. For detecting interacting proteins in the cytoplasm, the cytoplasmic fraction obtained by subcellular fractionation was used.
8
Immunoprecipitation and Immunoblotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 48 hr post-transfection of indicated plasmids, cells were harvested and lysed with radio-immunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% IGEPAL, 1 mM NaF, 1 mM Na3VO4) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma) and sonicated to prepare the whole cell lysate (WCL). WCL were precleared with Sepherose 6B (GE Healthcare Life Science) at 4°C at least for 2 hr. Precleared lysates were incubated with 50% slurry of glutathione-conjugated Sepharose (GST) beads (Amersham Biosciences) for GST pull-down, and for immunoprecipitation of anti-V5 and NLRX1, lysates were incubated with anti-V5 or NLRX1 antibody (1.0 μg/ml) for 12 hr and then protein A/G plus agarose beads (Santacruz) were added. Immunoprecipitates were collected by centrifugation, washed with lysis buffer in different washing conditions.
Additionally, WCL in control, FAF1 knockdown and overexpressing RAW264.7 and FAF1 knockdown and reconstituted MEFs infected with PR8-GFP during indicated time points were subjected to immunoblotting to analyze protein phosphorylation levels using respective antibodies.
Additionally, WCL in control, FAF1 knockdown and overexpressing RAW264.7 and FAF1 knockdown and reconstituted MEFs infected with PR8-GFP during indicated time points were subjected to immunoblotting to analyze protein phosphorylation levels using respective antibodies.
9
Flag-tagged Protein Immunoprecipitation for Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown on 15-cm plates and induced with doxycycline for 48 h. Agarose beads (Santa Cruz, #sc2003) were conjugated with anti-mouse IgG antibody (Santa Cruz, #sc2025) O/N at 4°C. NET-N (125mM NaCl, 50mM Tris-Hcl pH 8.0, 0.5% NP-40) buffer was used to lyse the cells and lysate was pre-cleared with Agarose beads (IgG antibody for 1 h at 4°C then beads for 30 min at 4°C). For Flag IP, lysate was incubated with flag-tagged beads (Biotool, #B23101) for 2 h at 4C. Beads were then washed with 1X TBS (as per protocol) and sent for mass spectrometry analysis to the proteomics core at UTMB. Samples were prepared in 5% SDS, 50mM TEAB, pH = 7.5. The Agarose beads were incubated for 30 min at 37°C, after which the supernatant was processed as described in the LC/MS section above.
10
Immunoprecipitation and Western Blot of RPA194
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The crude lysates were prepared with young adult animals in PRO-PREP lysis buffer (iNtRON, Seongnam, South Korea) supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland) by sonication. After centrifugation (11 000 × g, 4 °C, 10 min), the supernatants were collected and protein concentrations were determined by standard Bradford assay (Bio-Rad, Hercules, CA, USA). The lysate containing 2 mg of proteins was used to incubate with 4 μg of RPA194 antibody (sc-28714, Santa Cruz, Dallas, TX, USA) for 1–2 h at 4°C. The resulting immunocomplex was bound and precipitated with 50 μl protein A/G plus agarose beads (Santa Cruz) for 1 h. The beads were washed three times with NET buffer (50 mM Tris HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) and boiled in Laemmli sample buffer for 10 min. After 10 min of centrifugation (12 000 × g, 4°C), lysate suspensions of the respective sample were analyzed in 8% SDS-PAGE. Proteins were transferred to a PVDF membrane and the resulting blots were detected with the primary antibodies as indicated. The DAO-5-specific monoclonal antibody 5E9 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA)21 (link)was used in 5–10 times dilution with 5% skim milk/PBST (0.5% Tween-20). The blots were developed and detected with chemiluminescence ECL kit (T-Pro Biotechnologies, New Taipei City, Taiwan).
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!