Invitrogen recombinant rabbit polyclonal antibodies are unique offerings from Thermo Fisher Scientific. They are comprised of a mixture of recombinant antibodies co-expressed from a library of heavy and light chains. Recombinant rabbit polyclonal antibodies provide the best of both worlds–the sensitivity of polyclonal antibodies with the specificity of monoclonal antibodies–all delivered with the consistency only found in a recombinant antibody. While functionally the same as a polyclonal antibody, recognizing multiple epitope sites on the target and producing higher detection sensitivity for low abundance targets, the exact population of heavy and light chains in a recombinant polyclonal antibody can be produced in every lot, circumventing the biological variability typically associated with polyclonal antibody production.
Recombinant rabbit monoclonal antibodies are similar in consistency to traditional mouse monoclonal antibodies while offering an in vitro development option. Rabbits are immunized and, instead of using hybridomas, peripheral blood mononuclear cells (PBMCs) are isolated and screened. After the DNA is cloned as a library and screened, it is subcloned to a single clone and screened again. The resulting DNA is used for production of in vitro derived antibodies by cell culture.
Recombinant rabbit polyclonal antibodies exhibit superior lot-to-lot reproducibility because banked DNA does not exhibit cell drift–a phenomenon where hybridomas change expression patterns over time. Because the screening processes used in making recombinant rabbit polyclonal antibodies involves three different screening steps, the resulting antibody has better specificity and sensitivity than standard antibodies.
Figure 2. Immunofluorescence analysis of NFkB p65 was done on 70% confluent log phase ME-180 cells. The cells were either mock treated or treated with TNF-alpha (50 ng/mL for 20 minutes), fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were subsequently labeled with NFkB p65 (Green) Recombinant Rabbit Polyclonal Antibody (Cat. No. 710048) at 1µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034) at a dilution of 1:2,000 for 45 minutes at room temperature. Nuclei (Blue) were stained with SlowFade Gold Antifade Mountant DAPI (Cat. No. S36938). F-actin (Red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300). TNFalpha induced nuclear translocation of NFkB, a downstream target in the TNFR1, TRADD, and IKK alpha was observed in control cell line (panels a, e) and not in the TNFR1, TRADD, and IKK alpha knockout (KO) cell lines (panels b-d; f-h). The images were captured at 40X magnification.
Figure 3. Western blot analysis of Histone H3 (pS10) was performed by loading 20 µg of HeLa (lane 1) and HeLa treated for 25 minutes with 100 µM of Calyculin A (lane 2) cell lysates using Novex NuPAGE 4–12% Bis-Tris gel (Cat. No. NP0321BOX), XCell SureLock Electrophoresis System (Cat. No. EI0002), Novex Sharp Pre-Stained Protein Standard (Cat. No. LC5800), and iBlot Dry Blotting System (Cat. No. IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. Histone H3 (pS10) was detected at ~17 kDa using Histone H3 (pS10) Recombinant Rabbit Polyclonal Antibody (Cat. No. 710282) at 1 µg–3 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with phosphopeptide (10 µg/mL) as shown in corresponding blot on right. Goat anti-Rabbit IgG-HRP Secondary Antibody (Cat. No. G-21234) at 1:5,000 dilution was used, and chemiluminescent detection was performed using Pierce ECL Western blotting Substrate (Cat. No. 32106).
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