Rabbit CUP9 Polyclonal Antibody | anti-CUP9 antibody

Rabbit anti-Saccharomyces cerevisiae (strain 204508/S288c)(Baker's yeast) CUP9 Polyclonal Antibody

Cat. Num. AAA244196

• Reactivity: Saccharomyces cerevisiae (strain 204508/S288c)(Baker's yeast)
• Applications: Western Blot, ELISA
• Purity: Antibody purity > 90% confirmed by SDS-PAGE
• Synonyms: CUP9, Antibody; Rabbit anti-Saccharomyces cerevisiae (strain 204508/S288c)(Baker's yeast) CUP9 Polyclonal Antibody; CUP9 Antibody; Homeobox protein CUP9 CUP9 YPL177C; anti-CUP9 antibody

• Host: Rabbit
• Reactivity: Saccharomyces cerevisiae (strain 204508/S288c)(Baker's yeast)
• Clonality: Polyclonal
• Isotype: IgG
• Purity/Purification: Antibody purity > 90% confirmed by SDS-PAGE
• Form/Format: Liquid; Preservative: 0.03% Proclin 300; Constituents: 50% Glycerol, 0.01M PBS, pH7.4
• Sequence: MNYNCEIQNRNSKNVDNQVSLPPIQVLFNSIEKRSMPELAFSNIEYSHGNLRSSTEEQNYPAPVLLPQHHSIAYPAINSGGTSTTATPTASTVETSKTSSSAMDTQSQYGSSKKSKSASDDAKPCYKSAPIYEIINKEKDAGAQYNRPFSDFVESKSRRKQNSGRRSNL PKETVQILNTWLLNHLNNPYPTQQEKRELLIKTGLTKIQLSNWFINVRRRKIFSDYYTLVNSIPNDNANNTPVERVQNVSAYHNTLSATNNTMYDATSTCSTDYELSKRFAHAPVTRRKKLIDRLEELKKLSNPDMN

• Applicable Applications for anti-CUP9 antibody: WB (Western Blot), ELISA
• Immunogen: Recombinant Saccharomyces cerevisiae (strain 204508 / S288c) (Baker's yeast) CUP9 protein
• AA Range: 1-306aa
• Antigen Source: Escherichia coli (E. coli)
• Synthesis of Recombinant Antigen in Escherichia coli (E. coli) Cells: 1. Gene cloning - Insert gene of interest into a bacterial plasmid vector. This is typically done using DNA techniques like PCR, restriction digestion, and ligation.; 2. Transforming E. coli cells - Introducing the recombinant plasmid containing the gene of interest into chemically competent E. coli cells using a process like heat shock or electroporation.; 3. Selection of transformed cells - Plating the transformed cells on selective media to identify those containing the recombinant plasmid. Antibiotic resistance genes on the plasmid allow for selection.; 4. Small scale expression test - Growing selected colonies in small cultures to confirm expression of the recombinant protein, often detected by SDS-PAGE or Western blot.; 5. Optimization of expression conditions - Varying factors like inducer concentration, culture density, temperature to maximize yield.; 6. Large scale expression - Growing an optimized culture in large volumes to produce gram quantities of the recombinant protein.; 7. Cell lysis - Breaking open the cells by sonication or freeze-thaw cycles to release the protein.; 8. Protein purification - Purifying the target protein away from other cellular components using techniques like centrifugation, chromatography, precipitation etc.; 9. Analysis of purified protein - Assessing purity and molecular weight of the final product.
• Standard Rabbit Immunization Protocol: 1. Animal immunization: Two healthy Japanese white rabbits aged 3-6 months (1.5Kg-2Kg size) were selected.; 2. Adjuvant: Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA).; 3. Immunogen: Protein (6mg, purity>85%, concentration>1mg/ml), 500ug immunogen per immunization.; 4. Immunization: The immunogen was diluted with physiological saline and then mixed 1:1 with the corresponding adjuvant. The antigen and the adjuvant were completely mixed to form a stable emulsion. Extract the antigen mixture with a syringe and inject the antigen subcutaneously on both sides of the rabbit's shoulders, both hind legs, and both sides of the spine(6-8 points in total, about 1/3 volume of immunogen was injected per area), which allows the immunogen to persist and increase the immune response.; 5. Blood collection: The rabbits were bled from the ear artery with a 19-gauge needle and the serum was separated overnight at room temperature.; 6. 5 ml of whole blood (2-3 ml of pre-immune serum obtained) was allowed to stand at room temperature overnight, and was aliquoted after centrifugation in the next day. Perform ELISA screening test of the blank serum with antigen, two rabbits with no cross-reactivity were selected and subjected to immunization experiments.; Immunization 1: Rabbits were immunized with 500ug of antigen mixed with complete Freund's adjuvant (CFA);; Immunization 2: Rabbits were immunized with 500ug of antigen mixed with complete Freund's adjuvant (CFA);; Immunization 3: Rabbits were immunized with 500ug of antigen mixed with incomplete Freunds adjuvant (IFA);; Serum Collection 1: Collect 5 ml whole blood for ELISA detection;; Immunization 4: Rabbits were immunized with 500ug of antigen mixed with incomplete Freund's adjuvant (IFA). Start collecting serum if the ELISA produces positive results.; Serum Collection 2: Collect small sample (same as “Serum Collection 1”) if the ELISA result was not positive in Serum Collection 1. Samples were sent to the laboratory to be tested;; 7. Harvest the blood in the case of the blood meets requirement. The ELISA result of the test will determine the continuation, modification or termination of this project.; 8. Purification: Antigen affinity purification.; 9. Detection: Western blot validation with antigen
• ELISA Protocol: 1. Coat antigen at 2ug/ml(100ul per well) overnight under 4 degree C or 2 hours under 37 degree C; 2. Blocking the well with 5% non-fat dry milk for 2 hours under 37 degree C.; 3. Wash the well 3 times with TBS; 4. Perform a serial dilution for primary antibody (Table1.,100ul per well) and incubate for 1 hour under 37 degree C. Use PBS as the negtive control.; 5. Wash the well 3 times with TBS; 6. Dilute secondary antibody and incubate for 1 hour under 37 degree C.; 7. Wash the well 5 times with TBS; 8. Add TMB (90ul per well) and incubate for 5-20min minutes under 37 degree C (avoid light).; 9. Add Stop Solution (50ul per well) and read the optical density of each well using a microplate reader set to 450nm.
• WB Protocol: 1. Load 40ng, 20ng, 10ng and 5ng protein sample to the gel, 12% SDS-PAGE, 80V ~30min, 120V ~90min.; 2. Half-dry transfer: 60mA, 80min (0.45PVDF, Millipore).; 3. Block the membrane with 5% PBS and non-fat dry milk, incubate for 1h under 25 degree C.; 4. Dilute primary antibody with 0.05% PBST and 2.5% non-fat dry milk, and incubate overnight under 4 degree C.; 5. Wash the membrane with 0.1% PBST for 4 times*10min.; 6. Dilute secondary antibody HRP-Goat anti-rabbit IgG with 0.05% PBST and 2.5% non-fat dry milk, incubate for 2h at 25°C.; 7. Wash the membrane with 0.1% PBST for 4 times*10min.; 8. ECL chemiluminescence and X-ray imaging.
• Preparation and Storage: Upon receipt, store at -20 degree C or -80 degree C. Avoid repeated freeze.

WB (Western Blot)

ELISA

NCBI and Uniprot Product Information

• NCBI GI #: 6325080
• NCBI GeneID: 855926
• NCBI Accession #: NP_015148.1
• NCBI GenBank Nucleotide #: NM_001183991.1
• UniProt Accession #: P41817; D6W3J1
• Molecular Weight: 41.6 kDa
• NCBI Official Full Name: Cup9p
• NCBI Official Symbol: CUP9
• NCBI Protein Information: Cup9p
• UniProt Protein Name: Homeobox protein CUP9
• UniProt Gene Name: CUP9