The largest database of trusted experimental protocols

4 protocols using rabbit igg isotype control

1

ChIP-Seq Protocol for Histone Modifications

Check if the same lab product or an alternative is used in the 5 most similar protocols
The ChIP assay was performed according to company protocol (Upstate) with some modification. Cells were first fixed in 1% formaldehyde and then quenched by 125 mM glycine. Cells were collected and resuspended in 1% SDS lysis buffer, and then sonicated to shear DNA into 100–1000 bp fragments. The supernatant was collected and subjected to overnight immunoprecipitation with 4 µg H3K27ac antibody (Abcam, Cambridge, UK), 25 µg HDAC3 antibody (Abcam, Cambridge, UK), or rabbit IgG isotype control (Novus Biological, Littleton, CO, USA) as a mock control, together with Magna ChIP™ Protein A + G Magnetic Beads (Millipore, Burlington, MA, USA) at 4 °C in a rotatory shaker, followed by reverse cross-link and protease K digestion. Eluted DNA was then purified using a MinElute PCR Purification Kit (Qiagen, Hilden, Germany) and analysed by real-time PCR. Primer sequences are listed in Table 2.
+ Open protocol
+ Expand
2

Antibody Characterization for ESR1, ESR2, and GPER1

Check if the same lab product or an alternative is used in the 5 most similar protocols
The antibodies against ESR1 were rabbit polyclonal antibody HC-20 (against the C-terminus of the human protein) (sc-543, Santa Cruz Biotechnology, Inc. Heidelberg, Germany) and mouse monoclonal antibody MA1-310 (against the synthetic peptide within the DNA-binding domain of the human protein) (Thermo Fisher Scientific, IL, USA). The antibodies against ESR2 were rabbit polyclonal antibody H-150 (against amino acids 1–150 of the human protein) (sc-8974, Santa Cruz Biotechnology, Inc. Heidelberg, Germany) and mouse monoclonal antibody 6A12 MA1-23221 (against amino acids 1–153 of the human protein) (Thermo Fisher Scientific, IL, USA). The antibodies against GPER1 were rabbit polyclonal antibody K-19 (against the internal region of human GPR30) (sc-48524-R, Santa Cruz Biotechnology, Inc. Heidelberg, Germany) and rabbit polyclonal antibody H-300 (against amino acids 76–375 of human GPR30) (sc-134576, Santa Cruz Biotechnology, Inc. Heidelberg, Germany). The controls used in the experiments were rabbit IgG isotype control (Novus Biological, Centennial, CO, USA) and mouse IgG1/IgG2 isotype control (EXBIO, Vestec, Czech Republic).
+ Open protocol
+ Expand
3

Microinjection of Recombinant Proteins and Antibodies in MII Oocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols
MII oocytes were microinjected with 10 μM purified recombinant proteins (wild type α-SNAP, mutant α-SNAP L294A, wild type NSF and mutant NSF D1EQ) diluted in PBS if necessary. Anti-α-/β-SNAP, anti-γ-SNAP, and anti-NSF antibodies (Synaptic Systems) were microinjected at the maximum possible concentration. Anti-α-/β-SNAP, anti-γ-SNAP, mouse IgG isotype control (Novus Biologicals), and rabbit IgG isotype control (Novus Biologicals) were microinjected at 1 μg/μl. All antibodies and isotype controls were prepared in PBS. Microinjection pipettes were made by pulling borosilicate-glass capillary tubing in a mechanical puller (model P-97; Sutter Instrument Co., Novato, CA). Microinjections were performed using micromanipulators (Narishige) coupled to a Olympus IX-51 inverted microscope (Olympus). All micromanipulations were carried out in 5 μl drops of MEM/HEPES. For microinjection, needles were filled with injection solutions at the indicated concentrations, and about 7–10 pl was injected into the cytoplasm of MII oocytes by pneumatic pressure using a Pico-Injector (model PLI-100, Harvard Apparatus, Holliston, MA). Injected oocytes were used in CG exocytosis experiments after at least 1 h incubation in M16 medium, in a humidified atmosphere with 5% CO2 at 37°C. The number of oocytes used for each experiment is indicated in the figure legends.
+ Open protocol
+ Expand
4

ChIP Assay for Pparγ Binding

Check if the same lab product or an alternative is used in the 5 most similar protocols
The ChIP assay was performed as described previously [50 (link)]. The cells were subjected to overnight immunoprecipitation with 8 μg Pparγ antibody (Perseus Proteomics, Tokyo, Japan), or rabbit IgG isotype control (Novus Biological, Littleton, CO, USA) as a mock control and analyzed by real-time PCR. The primer sequences are listed in Table 2.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!