Immunoprecipitation and the HighSensitivity Protein 250 AssayCombining Specific and Sensitive Detection ofProteins with the Agilent 2100 Bioanalyzer

Abstract

A new method for the targeted analysis of proteins is presented that combines the

specificity of immunoprecipitation with the sensitivity of protein detection on

microchips using the High Sensitivity Protein 250 assay for the Agilent 2100

Bioanalyzer. As as an alternative to Western blotting, this method is valuable for

researchers engaged, for example, in protein expression and purification in pharma,

biotech or academic labs. Advantages of this new method in comparison to Western

blotting are:

• More reliable results: higher specificity and sensitivity

• Better accuracy and precision: less manual steps and direct availability of quantitative data

• Increased productivity: 3 hours versus 1 day analysis time

• Lower spending for antibodies: 10x less primary and no secondary antibody areneeded

• Lower reagent consumption: environmentally friendly process

Authors

Christian Wenz and Andreas Rüfer

Agilent Technologies, Inc.

Hewlett-Packard Strasse 8

76337 Waldbronn

Germany

Application Note

Protein Electrophoresis

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supplied with the High SensitivityProtein 250 kit. The eluted proteins aredirectly loaded onto microchips andanalyzed automatically with the Agilent2100 Bioanalyzer. Total time demand forthe IP/HSP250 method is about 3 h.Conventional immunoprecipitation tech-niques that use SDS-PAGE with silveror Coomassie staining for protein detec-tion, may have a problematic back-ground, because molecular tools likeantibodies and capture reagents arestained as well. This is not an issuewith the IP/HSP250 method since thesample proteins are labeled with a fluo-

Introduction

Today, immunoaffinity is a crucial toolfor the targeted analysis of proteins incomplex samples. Techniques likeEnzyme Linked Immunosorbent Assay(ELISA) and Western Blotting are wide-ly used for a broad range of applica-tions such as biomarker candidate veri-fication in body fluids or clone selectionfor recombinant protein expression. TheAgilent 2100 Bioanalyzer is a versatileanalytical tool that allows characteriza-tion of diverse biological samples suchas DNA, RNA, proteins and cells.1

However, most current applicationsmeasure all analytes in a given sizerange, for example the Protein 80 assaythat determines the size and quantity ofall sample proteins from 5 to 80 kDa.This Application Note describes anapplication for the targeted analysis ofproteins with the Agilent 2100Bioanalyzer based on the HighSensitivity Protein 250 assay in combi-nation with immunoprecipitation(IP/HSP250 method, Figure 1). TheHigh Sensitivity Protein 250 assayachieves protein sizing and quantifica-tion with sensitivity superior to silver-stained SDS-PAGE and a linear dynamicrange spanning up to four orders ofmagnitude.2 Figure 1 shows the work-flow of the IP/HSP250 method.Highlighted in yellow are steps of theHigh Sensitivity Protein 250 assay forsample preparation and final analysison chip.3 Reagents, microchips and pro-tocols necessary for these steps aresupplied with the High SensitivityProtein 250 kit. The immunoprecipita-tion was done after sample preparationemploying target specific antibodiesand Protein A coated magnetic beads.Elution of immunocomplexes from thebeads was done by heat denaturationin an SDS and DTT containing buffer

rescent dye in the initial sample prepa-ration step. Only these labeled speciesare detected after the immunoprecipita-tion by laser induced fluorescencedetection on-chip.

The performance of the IP/HSP250method is shown with E. coli cell lysatesamples spiked with a GST-tagged pro-tein and tag-specific antibodies. West-ern blots were prepared with the samesamples and antibodies to benchmarkthe new method against an establishedtechnique for targeted protein detection.

Labeling with the HSP250 fluorescent dye (45 min)

Pre-clearing with 1 µL Protein A magnetic beads (10 min)

Incubation with specific antibody (1 h)

Wash beads 3x with 200 µL buffer each (15 min)

Elution with water + 50 % sample buffer with DTT (5 min 95°C)

On-chip analysis with the HSP250 (30 min)

Incubation with 1 µL Protein A magnetic beads (10 min)

Protein sample(10 µL, 10 µg/µL)

Figure 1Workflow for the specific detection of tagged proteins with the IP/HSP250 method.

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ExperimentalMaterialDynabeads Protein A, DynaMag-2, XCell II Mini-Cell & Blot Module, pre-cast 4-12 % BisTris minigels, prestainedprotein standard, PVDF membrane,buffers for SDS-PAGE and Westerntransfer, WesternBreeze ChromogenicWestern Blot Detection Kit (Invitrogen,Carlsbad, CA, USA), Protein LoBindtubes (Eppendorf GmbH, Hamburg,Germany), Zeba Desalt Spin Columnsand Coomassie Plus Assay Reagent(Pierce, Rockford, IL, USA), lyophilizedE. coli strain B cells (ATCC 11303), rab-bit polyclonal anti-GST antibody (Sigma,Taufkirchen, Germany), GST-taggedPhosphatase and tensin homolog(PTEN), Agilent 2100 bioanalyzer andHigh Sensitivity Protein 250 kit (AgilentTechnologies GmbH, Waldbronn,Germany).

Sample preparationGST-tagged PTEN was supplied as 1 mg/mL solution in 50 mM Tris/HClpH 7.5, 50 mM NaCl, 1 mM EDTA and 5 mM DTT. Lyophilized E. coli cells wereresuspended in 50 mM sodium phos-phate buffer pH 8, 300 mM NaCl anddisrupted with a BeadBeater (BiospecProducts, Bartlesville, OK, USA). Celldebris was removed by centrifugation at12,000 g for 10 min. A buffer exchangeto 50 mM sodium phosphate buffer pH8.3, 300 mM NaCl was done with 2 mLZeba Desalt Spin Columns. Total proteinconcentration of the cell lysate wasdetermined with the Coomassie PlusAssay Reagent.

E. coli extract at a concentration of 10 µg/µL was spiked with 1 % of GST-tagged PTEN (corresponding to100 ng/µL target protein). Serial 10-folddilutions of this 1 % mixture with E. colilysate were prepared to obtain mixtureswith 0.1 % (10 ng/µL), 0.01 % (1 ng/µL),0.001 % (100 pg/µL) and 0.0001 % (10 pg/µL) tagged protein, respectively.Control samples of target protein (with-out E. coli lysate) were diluted withlabeling buffer (30 mM Tris/HCl, pH8.5) to 100 ng/µL.

Protein LabelingProtein labeling was done as describedin the Agilent High Sensitivity Protein250 Kit Guide.3

ImmunoprecipitationImmunoprecipitation was done inProtein LoBind tubes with 10 µL oflabeled sample. Tween 20 was addedto the sample to a final concentrationof 0.1 %.

Initially, samples were pre-cleared withProtein A beads to reduce background.One µL of magnetic Protein A beadswas washed twice with 100 µL of washbuffer (50 mM sodium phosphate, pH 8, 300 mM NaCl, 0.1 % Tween 20).Once washed, the beads were added tothe sample and incubated for 10 min onice. Tubes were vortexed every 2–3 minto keep the beads in suspension. After10 min, the cleared supernatant wascollected and the beads were dis-charged.

Second, samples were incubated withthe tag-specific antibody. Anti-GSTantibody was diluted 100 to 1 in wash

buffer. One µL of the antibody dilutionwas added to the sample and the mix-ture was incubated for 1 h on ice.

Next, immunocomplexes were capturedwith Protein A beads: One µL of ProteinA beads was washed twice with washbuffer and added to the sample. After10 min incubation on ice with vortexingevery 2–3 min, the supernatant was dis-charged. The beads were washed 3times with 200 µL wash buffer. Extracare was taken during the last washstep to remove the remaining liquidcompletely and avoid carry-over of salt.

Finally, captured proteins were elutedfrom the beads: The beads were resus-pended with 10 µL of water and 5 µL ofHigh Sensitivity Protein 250 samplebuffer with DTT to elute all bound pro-tein. After heating to 95 °C for 5 min,samples were centrifuged for 30 s andapplied directly (without dilution) to themicrochip.

On-chip analysisOn-chip sample analysis was done asdescribed in the Agilent High SensitivityProtein 250 Kit Guide.3 The HighSensitivity Protein 250 ladder waslabeled and analyzed according to thestandard procedure.

Western blotAccording to supplier instructions, SDS-PAGE was performed with 4 % to 12 %BisTris minigels. Western transfer wascompleted onto PVDF membranes.Finally, chromogenic immunodetectionwas completed with BCIT/NBT sub-strate and anti-rabbit secondary anti-bodies coupled to alkaline phosphatase.Primary anti-GST antibody was diluted,2000:1.

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Results and discussion

The sensitivity and specificity of theIP/HSP250 method was investigatedwith an E. coli lysate spiked with a dif-ferent amount of GST-tagged phos-phatase and tensin homolog (PTEN).Samples were labeled with a fluores-cent dye, and aliquots were analyzedwith the High Sensitivity Protein 250assay. No difference was observedbetween the samples (Figure 2A). Then,samples were immunoprecipitated witha polyclonal rabbit anti-GST antibodyand magnetic Protein A beads.Immunocomplexes eluted from thebeads were directly loaded ontomicrochips and analyzed with theAgilent 2100 Bioanalyzer. After subtrac-tion of the negative control run the LOD(S/N > 3) was determined to 0.001 % or100 pg/µL PTEN in a background of 10 µg/µL E. coli lysate (Figure 2B).Recovery of PTEN target protein out ofthe E. coli lysate was estimated toabout 15 % from the quantitative dataprovided by the Agilent 2100Bioanalyzer. The low backgroundobserved in the IP/HSP250 experimentsis in part due to the pre-clearing step(Figure 1). Therefore, sample pre-clear-ing can be helpful to further improvethe detection limit, especially for lowconcentrations of the target protein(data not shown). Electropherograms ofthe immunoprecipitated samplesappear very similar to those of the tar-get protein control, implying that no

Figure 2IP/HSP250 of E. coli lysate samples spiked with GST-tagged PTEN. (A) Analysis of samples after thelabeling step. Overlaid are electropherograms of samples with 0.1 % (red), 0.01 % (blue), 0.001 % (green),and a negative control without PTEN (light blue). A direct analysis of PTEN with the High SensitivityProtein 250 assay is shown in the inset (‘PTEN control’). (B) Analysis of samples after immunoprecipita-tion. Overlaid are electropherograms of samples with 0.1 % (red), 0.01 % (blue), 0.001 % (green), 0.0001 %(light blue), and a negative control without PTEN (magenta). The inset shows a zoom on the PTEN mainpeak of the 0.001 % sample (‘Zoom’).

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degradation or aggregation occurredduring immunoprecipitation (Figure 2).

Western Blots were prepared for com-parison. The same samples and tag-specific antibody were used as for theIP/HSP250 method (Figure 3). Blots forPTEN showed a high non-specific back-ground present in all lanes, which ispresumably due to the secondary anti-rabbit antibody used. A specific bandwas observed only at a concentration of1 % or 100 ng/µL PTEN in a back-ground of 10 µg/µL E. coli proteins(Figure 3, left panel). Thus, theIP/HSP250 method showed both highersensitivity and specificity as comparedto the Western blot, resulting in a 1,000fold lower limit of detection of the tar-get protein PTEN. Obviously, the risk ofpoor results due to low antibody qualityis lower for the IP/HSP250 method withonly a single antibody being used.Furthermore, the IP/HSP250 methodallows antibody cost savings since con-sumption of primary antibody is at least10 times lower than for Western blot-ting, and no secondary antibody isused. Additional advantages of theIP/HSP250 method over Western blot-ting are a significant faster time-to-result (3 h vs. one day), reduced manual Figure 3

Comparison of the IP/HSP250 method to Western blotting with chromogenic immunocomplex detec-tion for E. coli lysate samples spiked with GST-tagged PTEN. Sample volumes differed: 10 µL forIP/HSP250 vs. 6.5 µL for Western blotting. Shown are the Western blot (left) and the gel-like view of theHigh Sensitivity Protein 250 assay (right). The PTEN band is indicated with a red arrowhead. Molecularweights of standard proteins are shown (LM = lower marker).

Western blot6.5 µL

IP/HSP25010 µL

1 2 3 4 5 6 7 1 2 3 4 5 6 7

kDa

260

160

110

80

60

5040

30

20

kDa

240

150

95

63

45

2815

LM

1: 1 % PTEN2: 0.1 % PTEN3: 0.01 % PTEN4: 0.001 % PTEN

5: 0.0001 % PTEN6: E. coli only7: PTEN only

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intervention and the direct availabilityof quantitative data from the analysiswith the Agilent 2100 Bioanalyzer.

Conclusion

The new IP/HSP250 method for the tar-geted detection of proteins deliveredsuperior results in terms of specificity,sensitivity, usability, and time-to-resultas compared to conventional WesternBlotting with chromogenic immuno-complex detection. The method com-bines the specificity of immunoprecipi-tation with the high sensitivity ofmicrofluidic protein detection on theAgilent 2100 Bioanalyzer.

References

1. www.agilent.com/chem/labonachip

2.Performance characteristics of the Highsensitivity Protein 250 assay for theAgilent 2100 Bioanalyzer, TechnicalNote, 5989-8940EN, AgilentTechnologies.

3.Agilent High Sensitivity Protein 250 KitGuide, Agilent Technologies Manual,reference number G2938-90310.

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www.agilent.com/chem/2100-protein250

© Agilent Technologies, Inc., 2009, 2016 Published March 1, 2016Publication Number 5990-4097EN