Speedy/Ringo Antibody - BSA Free

Novus Biologicals | Catalog # NB100-2521

Novus Biologicals
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Key Product Details

Species Reactivity

Validated:

Human, Mouse, Rat, Sheep

Cited:

Human, Rat, Ovine

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Immunocytochemistry/ Immunofluorescence

Cited:

Western Blot, Immunocytochemistry/ Immunofluorescence, IF/IHC

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all Speedy/Ringo Primary Antibodies »

Product Specifications

Immunogen

A synthetic peptide made to a C-terminal region of human Speedy/Ringo (within residues 200-286). This immunogen is conserved in both isoforms 1 and 2. [Swiss-Prot Q5MJ69]

Reactivity Notes

Sheep reactivity reported in scientific literature (PMID: 19187565). Human reactivity reported in scientific literature (PMID: 24037419). Rat reactivity reported in scientific literature (PMID: 19075091).

Localization

Nuclear

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Scientific Data Images for Speedy/Ringo Antibody - BSA Free

Western Blot: Speedy/Ringo Antibody [NB100-2521]

Western Blot: Speedy/Ringo Antibody [NB100-2521]

Speedy-Ringo-Antibody-Western-Blot-NB100-2521-img0006.jpg
Western Blot: Speedy/Ringo Antibody [NB100-2521]

Western Blot: Speedy/Ringo Antibody [NB100-2521]

Speedy-Ringo-Antibody-Western-Blot-NB100-2521-img0005.jpg
Immunohistochemistry-Paraffin: Speedy/Ringo Antibody [NB100-2521]

Immunohistochemistry-Paraffin: Speedy/Ringo Antibody [NB100-2521]

Immunohistochemistry-Paraffin: Speedy/Ringo Antibody [NB100-2521] - Speedy/Ringo was detected in immersion fixed paraffin-embedded sections of human testis using Rabbit Anti-Human Speedy/Ringo polyclonal Antibody (Catalog # NB100-2521) at 1:300 for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC003). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the nuclei of sperm cells.
Western Blot: Speedy/Ringo Antibody [NB100-2521]

Western Blot: Speedy/Ringo Antibody [NB100-2521]

Western Blot: Speedy/Ringo Antibody [NB100-2521] - Detection of Speedy/Ringo in mouse testis (2ug/ml).
Speedy/Ringo Antibody

Western Blot: Speedy/Ringo Antibody [NB100-2521] -

Western Blot: Speedy/Ringo Antibody [NB100-2521] - Elevated Spy1 levels prevent contact inhibition. (A) Representative views of focus formation assays in NIH3T3 cells transfected with Myc-Spy1-WT, Myc-Spy1-TST, positive control Ras-V12 or empty vector PCS3 control. n = 3 (B) Quantification of the number of colonies. Error bars reflect SE between triplicate experiments. t test was performed;** P ≤ 0.01. n = 3 (right panel) Western blot analysis of one representative lysate from A-B. Image collected & cropped by CiteAb from the following publication (https://bmccancer.biomedcentral.com/articles/10.1186/1471-2407-12-45), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Speedy/Ringo Antibody

Western Blot: Speedy/Ringo Antibody [NB100-2521] -

Western Blot: Speedy/Ringo Antibody [NB100-2521] - Spy1 stable protein induces anchorage independent growth. (A) 293 cells were transfected with different concentrations of Myc-Spy1-WT, Myc-Spy1-TST or 30 μg of empty PCS3 vector control. Soft agar assay was carried out & plates photographed & quantified on day 14. Foci were averaged over 3 separate transfections for each experiment. n = 3 (B) Western blot analysis of one representative experiment; densitometry in lower panel. n = 3 (C) Representative foci after 14 day soft agar assay visualized using light microscopy. Ras-V12 is transfected as a positive control. n = 3. (D) Total numbers of foci were counted over 3 separate plates using separate transfections for each experiment. n = 3. Error bars reflect SE between triplicate experiments. t test was performed;** P ≤ 0.01. (E) Western blot analysis of experiments in Figure. 1C & D. Quantified using densitometry followed by normalization for Actin levels. (A-E) Error bars reflect SE between triplicate experiments. t test was performed;* P≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Image collected & cropped by CiteAb from the following publication (https://bmccancer.biomedcentral.com/articles/10.1186/1471-2407-12-45), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Speedy/Ringo Antibody

Immunocytochemistry/ Immunofluorescence: Speedy/Ringo Antibody [NB100-2521] -

Immunocytochemistry/ Immunofluorescence: Speedy/Ringo Antibody [NB100-2521] - CDC2 associates with SPDY at ERES. (A-C) 0 h GV stage oocyte labeled for CDC2 (A, green), DNA (A, blue), & SPDY (B, red). Both CDC2 & SPDY localize to the same cortical domain (C). Note that the center of this oocyte is dented (the area that contains the arrow) causing the structure to appear in the middle of the oocyte, whereas it is located in the cortex. (D-F) 0 h GV stage oocyte stained for Cyclin B (D, green), DNA (D, blue), & P-GM130 (E, red). Cyclin B did not localize to the P-GM130-labeled cortical domain (F). (G-I) 0 h GV stage oocyte labeled for PSTAIR (G, green), DNA (G, blue), & P-GM130 (H, red). Spatial overlap of PSTAIR & phosphorylated GM130 staining (yellow) in a cortical domain is evident in the merged image (I). Images are Z-projections of 2 (A-C) or 3 (D-I) consecutive sections; scale bars represent 20 μm in C, F, & I, & 5 μm in C', F', & I'. Arrows indicate the region of the oocyte that is shown enlarged in the insets (A'-I'). Arrowheads denote the position of the GV. Image collected & cropped by CiteAb from the following publication (https://bmcdevbiol.biomedcentral.com/articles/10.1186/1471-213X-9-8), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Speedy/Ringo Antibody

Western Blot: Speedy/Ringo Antibody [NB100-2521] -

Western Blot: Speedy/Ringo Antibody [NB100-2521] - Spy1 stable protein accelerates tumorigenesis in vivo.(A) Percentage of mice presenting with palpable tumors from 0-19 days post-transplant. Each data point represents 4 mice per indicated construct. The entire experiment was repeated three times using three independently derived overexpressing cell lines for each construct. Mann-Whitney Test was performed (p < 0.05). (A; lower blot) Western blots were conducted to measure the stability of Myc-Spy1-WT (left) & Myc-Spy1-TST (right) from representative time points. Empty vector control (Cntl) cell expression levels of Spy1 are seen in lane 1 of each blot. (B) Total tumor volume was calculated for both Spy1-HC11 (Spy1-WT) & Spy1-TST-HC11 (Spy1-TST) transplanted glands. Results were taken from 45 transplants using cells from 3 separate transfections. Error bars reflect SE between transplants from different transfections. Left hand panel reflects overall volume, right hand panel reflects volumes of tissues taken over a month post-transplant. Image collected & cropped by CiteAb from the following publication (https://bmccancer.biomedcentral.com/articles/10.1186/1471-2407-12-45), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Speedy/Ringo Antibody

Western Blot: Speedy/Ringo Antibody [NB100-2521] -

Western Blot: Speedy/Ringo Antibody [NB100-2521] - Spy1 protein levels are elevated in human breast cancers.(A) TMAs containing cores from invasive ductal carcinoma (IvDC), infiltrated ductal carcinoma (IfDC), intraductal carcinoma (IDC) & invasive lobular carcinoma (ILC) as well as pair-matched normal (PM-N) or cancer-free patients (CF-N) were analyzed for Spy1 expression. The Spy1 signal intensity was normalized to nuclear stain (TOTO-3/PI) signal. Patient numbers are indicated below the sample (N). (B) Breast normal & cancer cell lines were analyzed by western blot analysis. Actin was used as a loading control. One representative blot of 2. (C) Western blot of 3 replicate infections of pLKO control or pLKO Spy1 in MDA-231 cells. Middle panel represents the densitometry values over 3 separate experiments. Where Spy1 is corrected for actin levels. Lower panel reflects cell counts at 4, 6, & 24 h post-infection. (All panels) Data shown is mean ± s.d. Student's t-test was performed * P < 0.05;**P < 0.01. (D) Knockdown effects on cell counts of MCF7 cells over 72 h after transfection with either pSUPER empty vector (siCntl) or pSUPER-Spy1 (siSpy1) over 3 separate transfections. Data shown is mean ± s.d. Image collected & cropped by CiteAb from the following publication (https://bmccancer.biomedcentral.com/articles/10.1186/1471-2407-12-45), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Applications for Speedy/Ringo Antibody - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

reported in scientific literature (PMID 19187565)

Immunohistochemistry

reported in scientific literature (PMID 19187565)

Western Blot

2 ug/ml
Application Notes
In Western blot analysis, a band is seen at ~36 kDa (isoform 2). After a longer exposure, a 31 kDa band can be detected (isoform 1). There is also a non-specific band at ~58 kDa.

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

Tris-Citrate/Phosphate (pH 7.0 - 8.0)

Format

BSA Free

Preservative

0.1% Sodium Azide

Concentration

1.1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C. Do not freeze.

Background: Speedy/Ringo

Speedy/Ringo (Spy1) regulates the G1/S phase transition of the cell cycle by binding and activating CDC2, CDK2 and CDKN1B/KIP1. It mediates cell survival during the DNA damage process through activation of CDK2.

Alternate Names

hSpy/Ringo A, MGC110856, Rapid inducer of G2/M progression in oocytes A, RINGO A, Ringo3, SPDY1, speedy homolog 1 (Drosophila), speedy homolog A (Xenopus laevis), speedy protein A, speedy-1, Spy1, SPY1MGC57218

Gene Symbol

SPDYA

Additional Speedy/Ringo Products

Product Documents for Speedy/Ringo Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Product Specific Notices for Speedy/Ringo Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations for Speedy/Ringo Antibody - BSA Free

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Protocols

View specific protocols for Speedy/Ringo Antibody - BSA Free (NB100-2521):

Speedy/Ringo Antibody:
Western Blot Protocol

1. Perform SDS-PAGE (4-12%, Bis-Tris) on samples to be analyzed, loading 40 ug of total protein per lane.

2. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer apparatus.

3. Rinse membrane with dH2O and then stain the blot using ponceau S for 1-2 minutes to access the transfer of proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil.

4. Rinse the blot in TBS for approximately 5 minutes.

5. Block the membrane using 5% non-fat dry milk + 1% BSA in TBS, overnight at 4 degrees Celsius.

6. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.

7. Dilute the rabbit anti-speedy/ringo primary antibody (NB 100-2521) in blocking buffer and incubate 1 hour at room temperature.

8. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.

9. Apply the diluted rabbit-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) and incubate 1 hour at room temperature.

10. Wash the blot in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each (this step can be repeated as required to reduce background).

11. Apply the detection reagent of choice in accordance with the manufacturer's instructions (Pierce's ECL).

Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%, provided it does not interfere with antibody-antigen binding.

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