PI3K p85 alpha/gamma antibody (pTyr199, pTyr467)

Catalog No. ABIN744743

Product Details anti-PI3K p85 alpha/gamma Antibody

Target: PI3K p85 alpha/gamma (PI3K p85a/g) (Phosphoinositide 3 Kinase, p85 alpha/gamma (PI3K p85a/g))

Binding Specificity: pTyr199, pTyr467

Reactivity: Human, Mouse, Rat, Rabbit

Host: Rabbit

Clonality: Polyclonal

Conjugate: This PI3K p85 alpha/gamma antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Flow Cytometry (FACS), Immunofluorescence (Cultured Cells) (IF (cc)), Immunofluorescence (Paraffin-embedded Sections) (IF (p)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunohistochemistry (Frozen Sections) (IHC (fro))

Cross-Reactivity: Human, Mouse, Rabbit, Rat

Predicted Reactivity: Dog,Cow,Pig,Horse

Purification: Purified by Protein A.

Immunogen: KLH conjugated synthetic phosphopeptide derived from human PI3K around the phosphorylation site of p85 alphaTyr467

Isotype: IgG

Application Details

Application Notes: WB 1:300-5000
ELISA 1:500-1000
FCM 1:20-100
IHC-P 1:200-400
IHC-F 1:100-500
IF(IHC-P) 1:50-200
IF(IHC-F) 1:50-200
IF(ICC) 1:50-200

Restrictions: For Research Use only

Independent Validation (WB)

Validation #029801 (Western Blotting)

by: Alamo Laboratories Inc

No.: #029801

Date: 08/26/2014

Lot Number: 120522

Method validated: Western Blotting

Positive Control: HeLa G2/M Phase cells

Negative Control: C6/36 cells (non-reactive species)

Notes: A strong band was observed in the positive control sample at the correct molecular weight, this band is absent from the negative control. Several bands were observed in the negative control sample which may represent non-specific binding.

Validation Images

Figure 1: Western Blot for PI3K p85 /p55 (pTyr467). Arrowhead indicates the expected molecular weight of ~85 kDa.

Full Methods

Primary Antibody:

• Antigen: PI3K p85 /p55 (pTyr467)
• Catalog number: ABIN744743
• Lot number: 120522
• Antibody Dilution: 1:200

Secondary Antibody:

• Antigen: Goat Anti-Rabbit IgG (H + L)-HRP Conjugate
• Lot number: L170-6515
• Antibody Dilution: 1:10,000

Full Protocol:

• 1. The cell extracts were heated at 95°C for 5 minutes in 1X SDS Sample Buffer containing 1% SDS and 1.25% β-mercaptoethanol.
• 2. 18 μl of heated culture-media were loaded and resolved on 8-16% SDS-polyacrylamide gel.
• 3. The Thermo Scientific - Spectra Multicolor Broad Range (Cat # 26634) were used as molecular mass markers.
• 4. Proteins were then transferred onto PVDF membrane by wet transfer and protein transfer was confirmed with Ponceau-S staining.
• 5. The PVDF membrane was incubated with 25 ml of blocking buffer [Tris Buffered Saline, pH 7.4 plus 0.1% TW20 (TBST)] containing 5% (W/V) BSA at room temperature for 1 hour.
• 6. The membrane was rinsed with TBST once.
• 7. The membrane was immersed with the protein side up in the primary antibody solution in TBST containing 5% (W/V) BSA and incubated for 10 hours at 4°C.
• 8. The membrane was rinsed in TBST thrice for 5 minutes each.
• 9. The membrane was incubated in the HRP-conjugated secondary antibody solution in TBST containing 5% (W/V) BSA and incubated for 1 hour at room temperature (~26°C) with gentle agitation.
• 10. The membrane was rinsed thrice TBST thrice for 5 minutes each.
• 11. The membrane was rinsed in TBS twice for 30 seconds each.
• 12. Signals were detected with ECL-2 Substrate. The blot was scanned for 15 minutes.
• 13. The membrane was rinsed three times TBST.
• 14. Incubated in Acidic Glycine Stripping Buffer at room temperature with gentle agitation for 3 times, 10 minutes each.
• 15. The membrane was washed in TBST 2 times for 10 minutes each.
• 16. Repeated Steps 5-12 with the loading control antibody (for Anti-actin) and its matching secondary antibody.

Experimental Notes: - No experimental challenges noted.

Handling

Format: Liquid

Concentration: 1 μg/μL

Buffer: 0.01M TBS( pH 7.4) with 1 % BSA, 0.02 % Proclin300 and 50 % Glycerol.

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.

Expiry Date: 12 months

References for anti-PI3K p85 alpha/gamma antibody (ABIN744743)

Jeong, Liu, Kim: "Dried plum and chokeberry ameliorate d-galactose-induced aging in mice by regulation of Pl3k/Akt-mediated Nrf2 and Nf-kB pathways." in: Experimental gerontology, Vol. 95, pp. 16-25, (2017) (PubMed).

Padiya, Chowdhury, Borkar, Srinivas, Pal Bhadra, Banerjee: "Garlic attenuates cardiac oxidative stress via activation of PI3K/AKT/Nrf2-Keap1 pathway in fructose-fed diabetic rat." in: PLoS ONE, Vol. 9, Issue 5, pp. e94228, (2014) (PubMed).

Sun, Yang, Luo, Wang, Chen, Zhang, Wang, Li: "Thyroid hormone inhibits the proliferation of piglet Sertoli cell via PI3K signaling pathway." in: Theriogenology, (2014) (PubMed).

Target Details for PI3K p85 alpha/gamma

Target: PI3K p85 alpha/gamma (PI3K p85a/g) (Phosphoinositide 3 Kinase, p85 alpha/gamma (PI3K p85a/g))

Alternative Name: PI3 kinase p85 alpha + gamma + (PI3K p85a/g Products)

Background:

Synonyms: p85, AGM7, GRB1, IMD36, p85-ALPHA, Phosphatidylinositol 3-kinase regulatory subunit alpha, PI3-kinase regulatory subunit alpha, PI3K regulatory subunit alpha, PtdIns-3-kinase regulatory subunit alpha, Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha, PI3-kinase subunit p85-alpha, PtdIns-3-kinase regulatory subunit p85-alpha, PIK3R1

Background: Binds to activated (phosphorylated) protein-Tyr kinases, through its SH2 domain, and acts as an adapter, mediating the association of the p11 catalytic unit to the plasma membrane. Necessary for the insulin-stimulated increase in glucose uptake and glycogen synthesis in insulin-sensitive tissues. Plays an important role in signaling in response to FGFR1, FGFR2, FGFR3, FGFR4, KITLG/SCF, KIT, PDGFRA and PDGFRB. Likewise, plays a role in ITGB2 signaling.

Gene ID: 5295

UniProt: P27986