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Demonstration of Western Blot Specific Antibody Production

Left panel: Alpha-fold predicted structure of mouse ENaC alpha protein. The antigen epitope is from the N-terminus. The epitope is denatured, stabilized and immunized to generate antibody. Right panel: HEK293 cells were transfected with mouse ENaC alpha subunit, which has a V5 tag at the C-terminus. A negative control (untransfected HEK293 cell lysates) and a positive control (transfected HEK293 cell lysates immunoprecipitated by V5 antibody) were included. All lysates were blotted with anti-ENaC alpha Western blot specific antibody. The Western blot specific antibody recognizes a specific band in the HEK+ ENaC lysates, which is the same size as the V5 IP protein, indicating the correct size for ENaC alpha protein. (Data kindly provided by Dr. Shujie Shi, University of Pittsburgh, Dept of Nephrology)

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Western Blot Specific Antibody Production

$2,150.00 – $8,950.00

Antibody: Polyclonal and Monoclonal Antibody

Concentration: 0.25 mg/ml of purified IgG

Application: Western Blot

Reactivity: Human, Mouse, Rat

BiCell Scientific Inc has developed Western blot-specific recognition antibodies with novel denaturing and stabilizing technique to prevent the natural folding of epitope sequence in order to lock the conformational state of epitope in unfolded state.

Western Blot detects denatured and linearized proteins in SDS buffers. Conventional antibodies raised against epitopes assuming natural folding conformations are not suited for Western Blot. When used for Western Blot, conventional antibodies often report multiple bands of different molecular weights but only one band being specific binding.

BiCell Scientific Inc has developed a novel denaturing and stabilizing technique to prevent the natural folding of epitope sequence in order to lock the conformational state of epitope in unfolded state. BiCell Scientific Inc is able to raise polyclonal and monoclonal antibodies against the unfolded target epitope. Such antibodies can dramatically improve the binding specificity on Western Blot, which will be vital tools of quantitative measurements of protein expression in endogenous cell or tissue samples.

Polyclonal Antibody Workflow Chart

StepsTimeline (weeks)Deliverables/Internal testing milestoneQC standard
Phase I: Antigen preparation

(peptide antigen,13-19 aa length, will be designed against target protein, synthesized, denatured, stabilized and conjugated to KLH)

2 weeks/COA

 

 

 

 

Phase II: Animal immunization

(3 Sprague Dawley rats or 5 Swiss Webster mice or 2 New Zealand rabbits will be immunized with antigen; conventional protocol – three injections)

 

 

8-10 weeks100 ul antiserumELISA after 3rd injection
Phase III: Antiserum collection

(antiserum will be collected from one best responder animal according to ELISA report)

 

 

1-2 weeks1 ml to 50 ml antiserumELISA from production bleed
Phase IV: Affinity Chromatography and Depletion Chromatography

(antiserum will be purified with affinity column conjugated with the antigen peptide)

 

1-2 weeks1 mg of purified polyclonal IgGWestern Blot

Monoclonal Antibody Workflow Chart

StepsTimeline (weeks)Deliverables/Internal testing milestoneQC standard
Phase I: Antigen preparation

(peptide antigen,13-19 aa length, will be designed against target protein, synthesized, denatured, stabilized and conjugated to KLH)

2 weeks/COA

 

 

 

 

Phase II: Animal immunization

(3 Sprague Dawley rats or 5 Swiss Webster mice or 2 New Zealand rabbits will be immunized with antigen; conventional protocol – three injections)

 

 

8-10 weeks100 ul anti-serumELISA after 3rd injection
Phase III: Cell fusion and screening

(fusion of mouse myeloma cells with splenocytes from one best responder animal to generate hybridoma and perform antigen based hybridoma selection, enriching, and plating)

 

 

4-6 weeksSupernatant from 96 wellsELISA from supernatant
Phase IV: Expansion and antibody purification

(customer choosing one clone; expanding clone and purifying antibodies with affinity chromatography)

 

6-8 weeksOne hybridoma cell line (to customer); purified monoclonal antibody from the selected hybridoma cell line (1mg)

(BiCell Scientific will bank all remaining clones; customer can request additional clones by paying a service fee for hybridoma recovery)

Western Blot

 

For Research Use Only. Not for use in clinical diagnostics.

Additional information

Antibody Deliverable

1 mg of Western Blot Specific Polyclonal Antibody Production, 1 mg of Western Blot Specific Monoclonal Antibody Production

Host Species

Rat, Mouse, Rabbit

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"I am really impressed with your approach. We tried multiple times previously to create monoclonal and polyclonal antibodies to claudin-2 and MLCK1. We have had limited success generating polyclonals and no success generating monoclonals. You have generated outstanding monoclonals to both. I look forward to continuing to work with you."

Jerrold R. Turner, M.D., Ph.D.

Brigham and Women’s Hospital | Harvard Medical School

"The polyclonal antibody you generated for KIAA0408 is stunning! KIAA0408 is a novel cilium molecule that has never been studied. So, clearly there will be a lot of demand for it as we have discovered a very interesting finding and the story will be published in a high impact journal. I am strongly inclined to generate monoclonal antibody for this protein too and we should think about patenting it."

Univ.-Prof. Jay Gopalakrishnan PhD

Heinrich-Heine-Universität | Universitätsklinikum Düsseldorf

"Your ARL13B antibody works beautifully!!! We’re so happy to have a cilia-specific antibody made in rat! I can send you high resolution images to be posted on your website."

Julie Craft Van De Weghe, PhD

School of Medicine | University of Washington

"The assay is a homophilic interaction mediated cell adhesion on purified protein (in this case, immobilized purified Pcdhga9 to Pcdhga9 expressed on cell surface). Compared to control, cell adhesion is reduced in the presence of Pcdhga9 monoclonal antibody supernatants!"

Divyesh Joshi, PhD

School of Medicine | Yale University

I have tested the rat polyclonal IgGs to ABCD1 by immunoflourescence on cells overexpressing ABCD1. The antibodies successfully detected the protein (either untagged or tagged with GFP) at a 1:500 dilution and there was little background. The antibodies did not detect ABCD2. So this is very good news, and you may now go ahead and clone out a monoclonal!

Annette Ehrhardt, PhD

Dept. of Pediatrics | Emory University

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