Analytical Biochemistry 404 (2010) 244–246

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Analytical Biochemistry

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Notes & Tips

A visual chip-based coimmunoprecipitation technique for analysisof protein–protein interactions

Qing Chen a,1, Qiongming Liu a,1, Zhoumin Li b, Wenying Zhong b, Wei He a,**, Danke Xu a,c,*

a State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, Chinab Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, Chinac Key Laboratory of Analytical Chemistry for Life Science (MOE), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 April 2010Received in revised form 5 May 2010Accepted 6 May 2010Available online 10 May 2010

Keywords:Protein–protein interactionsSilver enhancement detectionProtein microarraysCoimmunoprecipitation

0003-2697/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.ab.2010.05.003

* Corresponding authors at: key Laboratory of AScience (MOE), School of Chemistry and Chemical Eng22 Hankou Road, Nanjing 210093, China. Fax: +86-10** Corresponding authors.

E-mail addresses: hewei1012@163.net (W. He), xud1 These authors contributed equally to this work.2 Abbreviations used: PPI, protein–protein interactio

tion; vChip–coIP, visual chip-based coIP; IgG, immunoated bovine serum albumin; HEK293, human embDulbecco’s modified Eagle’s medium; FBS, fetal boviaminetetraacetic acid; PMSF, phenylmethanesulfonyl fl

Here we report a visual chip-based coimmunoprecipitation (vChip–coIP) platform for analysis of protein–protein interactions (PPIs) by combining advantages of an antibody microarray, traditional coIP, and asilver enhancement detection method. The chip was fabricated by spotting anti-Flag antibody on alde-hyde-modified slides, and the resulting platform could assay immunoprecipitate from a small amountof crude cell lysates containing Flag–bait and Myc–prey. The interaction signals are visible using biotin-ylated anti-Myc antibody and colloidal gold-labeled streptavidin followed by a silver enhancement detec-tion method. It is shown that vChip–coIP is a simple, cost-effective, and highly efficient platform for thecomprehensive study of PPIs.

� 2010 Elsevier Inc. All rights reserved.

The human genome consists of 20,000 to 30,000 genes encod-ing more than 500,000 different proteins. Moreover, a cell can pro-duce more than 10,000 proteins at any given time. It has beenestimated that more than 80% of proteins do not operate alonebut rather operate in complexes by which protein–protein interac-tions (PPIs)2 are regulated by several mechanisms [1–3]. Most, ifnot all, biological processes require the cooperation of at least twoproteins and may require additional proteins for highly complexfunctions. Therefore, the analytical methods of PPIs, such as coim-munoprecipitation (coIP), affinity chromatography, and two-hybridassays, are essential for the elucidation of biological processesand/or networks [4–6]. However, the traditional resin-based coIPis time-consuming, especially for high-throughput analysis. Morerecently, protein arrays have been developed to assay PPIs [7–9],and several detection strategies based on fluorescent or radiometriclabels have been compared [10].

ll rights reserved.

nalytical Chemistry for Lifeineering, Nanjing University,-80705199.

k@nju.edu.cn (D. Xu).

n; coIP, coimmunoprecipita-globulin G; Bio-BSA, biotinyl-ryonic kidney 293; DMEM,

ne serum; EDTA, ethylenedi-uoride.

In this article, we report a visual chip-based coIP (vChip–coIP)technology for rapid analysis of PPIs, and its principle is illustratedin Fig. 1A. The protein array chip was fabricated by spotting anti-bodies with the Smart Arrayer 48 Spotting Robot (CapitalBio, Bei-jing, China) on CSS aldehyde-modified slides (CEL Associates,Pearland, TX, USA). Here 0.2 mg/ml murine immunoglobulin G(IgG), 1 mg/ml anti-Flag M2 antibody (Sigma, St. Louis, MO, USA),and 0.02 mg/ml biotinylated bovine serum albumin (Bio-BSA) werespotted from left to right, respectively (Fig. 1B). All slides wereincubated overnight at 4 �C to allow maximum binding of antibod-ies to the aldehyde slides, followed by blocking with 10 mg/ml BSAin TBST (20 mM Tris–Cl [pH 8.0], 150 mM NaCl, and 0.05% [v/v]Tween 20) at room temperature for 1 h. After blocking, the slideswere rinsed three times with TBST, followed by adding cell lysateto the slide to analyze protein expression and concentration.

To study cell lysate protein expression and concentration on PPIsignals, two cell lysates with differing antibodies were employed.First, Flag–p50 and c-Myc–p65 were used as positive controls,and Flag–Jun and c-Myc–lacZ were used as negative controls.Human embryonic kidney 293 (HEK293) cells were cultured inDulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad,CA, USA) supplemented with 10% fetal bovine serum (FBS, HyClone,Logan, UT, USA), penicillin, streptomycin, and glutamine. Transfec-tions were performed with Lipofectamine 2000 (Invitrogen) accord-ing to the manufacturer’s instructions. After 30 h, cells wereharvested and lysed in EBC buffer (50 mM Tris–Cl [pH 8.0], 120mM NaCl, 0.5% [v/v] NP40, and 1 mM ethylenediaminetetraaceticacid [EDTA]) supplemented with 50 lg/ml phenylmethanesulfonyl

Fig. 1. (A) Principle and process of the vChip–coIP technique. (B) Pattern of spotting antibody: (1) murine IgG (as negative spots), (2) anti-Flag M2 antibody, and (3) Bio-BSA(as positive spots). (C) Scanned images of the interacting protein pairs of Flag–p50 and c-Myc–p65 (positive control) and Flag–Jun and c-Myc–lacZ (negative control). Proteinconcentrations were 0, 0.15, 0.30, 0.55, 1.10, 2.25, and 4.50 lg/ll (from left to right). (D) Relationship between the gray levels and protein concentrations of cell lysate. j,Flag–p50 and c-Myc–p65; N, Flag–Jun and c-Myc–lacZ.

Notes & Tips / Anal. Biochem. 404 (2010) 244–246 245

fluoride (PMSF) and protease inhibitor cocktail (Roche, Basel,Switzerland) at room temperature for 10 min. After centrifugation(4 �C, 12,000 rpm, 10 min), the protein concentration of the superna-tant was determined by the Bradford method. Finally, 20 ll of cell ly-sate was transferred into detection wells of the chip and incubatedfor 2 h at room temperature.

The chips were washed three times with TBST to remove un-bound proteins. The interacting partners on the microarrays wereincubated with 0.02 mg/ml biotinylated monoclonal anti-c-Mycantibody (Sigma) in a humid chamber for 1 h. The excess unboundanti-c-Myc antibody was removed, followed by washing with TBST.Colloidal gold-labeled streptavidin (Sigma) and substrate silver en-hancer solution (Sigma) were added to incubate with the interact-ing partners sequentially for the desired time. Finally, the slideswere rinsed thoroughly with water to remove the excess substrate

solution, followed by scanning using a dual-media scanner (Scan-Maker 8700, Microtek, China). The scanned images (Fig. 1C) revealblack spots on the anti-Flag M2 antibody for the samples of the po-sitive control, whereas the negative control samples containingFlag–Jun and c-Myc–lacZ do not have black spots, thereby confirm-ing the positive and negative controls, respectively. In addition, themurine IgG spots do not show any dark images, suggesting thatthere is a low cross-reactivity performance on the arrays and theinteracting protein pair could be visualized by this antibodychip-based method.

To further extract the gray values of the spots, the images of theantibody arrays were processed with Spot Data Pro 2.0 (Capital-Bio), and the relative data are shown in Fig. 1D. Surrounding thegray values is the background, which was subtracted from theanti-Flag antibody spots. The protein pair of Flag–p50/c-Myc–p65

Fig. 2. Evaluation of 5 noninteracting protein pairs (1: Jun/c-Term1; 2: Jun/Jlip; 3:Jun/c-Term2; 4: Jun/DPH; 5: Jlip/LZPR) and 5 well-known interacting protein pairs(6: TRB3/ATF4; 7: Mafk/Nrf2; 8: Keap1/Nrf2; 9: MafG/Nrf2; 10: PLK1/TANK) by thevChip–coIP method. HEK293 cells were cotransfected with Flag–bait and Myc–preyconstructs, and 1 lg/ll cell lysates were used.

246 Notes & Tips / Anal. Biochem. 404 (2010) 244–246

was detectable at 0.25 lg/ll. Moreover, the cell lysate protein sig-nals increased proportionally with the increase in protein concen-tration, followed by signal saturation at 1.0 lg/ll, whereas thenegative controls of Flag–Jun and c-Myc–lacZ maintained ratherlow gray values. These results indicate that the strength of inter-acting protein signals was dependent on the protein concentrationof cell lysate below 1.0 lg/ll. To avoid this influence, the proteinconcentration of 1.0 lg/ll in cell lysate was employed in the fol-lowing analysis.

Furthermore, the concentration of biotinylated anti-c-Myc anti-body and silver enhancement reaction time are crucial experimen-tal parameters that affect the intensity of the final signal [11]. Inour experiments of optimizing analytical parameters, it can befound that the gray levels of the spots increase with the concentra-tion of the antibody and then level off at the concentration of20 lg/ml. The relationship of signal intensity with silver enhance-ment time indicates that the silver enhancement approaches satu-ration and the gray levels reached a plateau at 18 min, at whichpoint the detection gives the best ratio of signal to noise. Thus,the concentration of 20 lg/ml biotinylated anti-c-Myc antibodyand 18 min of silver enhancement time were employed for the fol-lowing measurements.

Based on the above optimized conditions, this method was fur-ther used to evaluate 5 pairs of noninteracting protein (Jun/c-Term1, Jun/Jlip, Jun/c-Term2, Jun/DPH, and Jlip/LZPR) and 5 pairsof well-known interacting protein (TRB3/ATF4, Mafk/Nrf2, Keap1/Nrf2, MafG/Nrf2, and PLK1/TANK). The experiment was repeatedthree times, and the values of the gray levels on the chips were cal-culated according to the above method. The relative statisticalanalysis was processed, and analysis of variance was used to eval-uate the method’s reproducibility. The data showed no significantdifference for the 5 pairs of noninteracting protein pairs and the 5pairs of well-known interacting protein pairs (P = 0.9880 and

P > 0.05, respectively). These results suggest that the method isboth accurate and reproducible.

As seen in Fig. 2, 5 pairs of noninteracting protein produced novisible spots, and the average of gray values was 41.8. To evaluatewhether there is an interacting protein pair based on its gray value,the cutoff value was calculated as the average value plus doublethe standard error. Based on this principle, the cutoff value wasdetermined to be 162.5. The gray values of the well-known inter-acting protein pairs were assayed to be 609.7 (TRB3/ATF4), 511.3(Mafk/Nrf2), 411.3 (Keap1/Nrf2), 762.0 (MafG/Nrf2), and 4591.1(PLK1/TANK). They are much higher than the cutoff value. As aresult, this method could provide a novel and simple assessmentfor PPI.

Compared with traditional resin-based coIP, vChip–coIP re-quires much smaller amounts of cell lysate and allows a large num-ber of samples to be studied en masse for their PPI informationwithout the need for further manipulation of the slides such asimmunofluorescence staining or enzymatic amplification. Our re-sults indicate that vChip–coIP could provide a highly accurate,cost-effective, and highly efficient platform in practical applica-tions to assay PPIs.

Acknowledgments

The authors acknowledge financial support from theNational Natural Foundation of China (20975050, 20575079) andthe National Basic Research Program of China (973 Program,2006CB910803).

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