纳流体蛋白质组免疫分析

Huifang Guo; Philip L Lorenzi; Zhiyong Ding

doi:10.21769/BioProtoc.1537

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Peer-reviewed

Nanofluidic Proteomic Immunoassay

纳流体蛋白质组免疫分析

HG Huifang Guo

Philip L Lorenzi

ZD Zhiyong Ding email

发布: 2015年07月20日第5卷第14期 DOI: 10.21769/BioProtoc.1537 浏览次数: 8331

评审: Pia GiovannelliAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Oncogene, featuring study using the protocol.

Jun 2014

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Abstract

Nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, has the potential to quantitatively characterize protein post-translational modifications that cause shifts in isoelectric point (pI). This protocol details the NIA analysis of protein phosphorylation using AKT as an example.

This protocol can be used for two platforms, NanoPro 1000 and Peggy Sue, from ProteinSimple Company. NanoPro 1000 separates proteins based on charge. Peggy Sue separates proteins based on either charge or size. The platforms can analyze up to 96 samples at a time with lysates from as few as 25 cells per assay. Detailed information of the platforms is available on ProteinSimple’s website (http://www.proteinsimple.com).

Keywords: Immunoassay (免疫分析法)

Antibody (抗体)

AKT (Akt)

NIA (NIA)

Phosphorylation (磷酸化)

Materials and Reagents

Reagents from ProteinSimple
1. 25x aqueous protease/phosphatase inhibitor mix (ProteinSimple, catalog number: 040482 )
2. 50x DMSO protease/phosphatase inhibitor mix (ProteinSimple, catalog number: 040510 )
3. Premix G2 (Servalyt pH 5-8 separation gradient) (ProteinSimple, catalog number: 040974 )
4. Premix G2 (pH 5-6 separation gradient) (ProteinSimple, catalog number: 040971 )
5. PI standard 5.5 (ProteinSimple, catalog number: 040028 )
6. PI standard ladder 3 (ProteinSimple, catalog number: 040646 )
7. Anolyte refill (ProteinSimple, catalog number: 040337 )
8. Catholyte refill (ProteinSimple, catalog number: 040338 )
9. Wash concentrate refill (ProteinSimple, catalog number: 041108 )
10. Amplified rabbit secondary antibody detection kit (ProteinSimple, catalog number: 041126 )
11. Peroxide XDR (ProteinSimple, catalog number: 041084 )
12. Amplified rabbit secondary antibody detection kit (includes SA-HRP conjugate, secondary goat-anti-rabbit-biotin conjugate and antibody diluent) (ProteinSimple, catalog number: 041126 )
13. RIPA lysis buffer (ProteinSimple, catalog number: 040483 ) (see Recipes)
14. Bicine/CHAPS lysis buffer and sample diluent (ProteinSimple, catalog number: 040764 ) (see Recipes)
15. Anolyte (10 mM Phosphoric Acid) (see Recipes)
16. Catholyte (100 mM Sodium Hydroxide) (see Recipes)
Antibodies for AKT analysis
1. AKT (pan) antibody (Cell Signaling Technology, catalog number: 4691 )
2. AKT1 antibody (BD Biosciences, catalog number: 610861 )
3. AKT2 antibody (Cell Signaling Technology, catalog number: 3063 )
4. Phospho-AKT (Thr308) (Cell Signaling Technology, catalog number: 2965 )
5. Phospho-AKT (Thr450) (Cell Signaling Technology, catalog number: 9267 )
6. Phospho-AKT (Ser473) (Cell Signaling Technology, catalog number: 9271 )
Other materials
1. BCA protein assay kit (Pierce Antibodies, catalog number: 23225 )
2. Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5 g/L glucose & L-glutamine without pyruvate (Mediatech, catalog number: 10017CV )
3. McCoy’s 5A (1x, with L-glutamine) (Mediatech, catalog number: 10050CV )
4. Fetal bovine serum (FBS) (Life Technologies, Gibco^®, catalog number: 10438026 )

Equipment

NP1000 or Peggy (ProteinSimple, model: 004800 )
Centrifuge for microplates (Thermo Fisher Scientific, model: Sorvall Legend XTR )
Capillaries Charge Separation (ProteinSimple, catalog number: CBS701 )
Assay Plate/Lid Kit (ProteinSimple, catalog number: 040663 )
Sponge Pack (ProteinSimple, catalog number: 041528 )
Tissue culture dish (100 mm) (Corning Incorporated, catalog number: 430167 )
Microcentrifuge tube (Denville Scientific, catalog number: C2170 )