Reverse Phase Protein Array – a high throughput proteomic tool

Discover the potential of RPPA and its many advantages over other proteomic techniques

Reverse Phase Protein Array (RPPA) is an antibody-based high throughput proteomic approach that enables the simultaneous quantification of large sample sizes (up to 1500 samples at once). Examples of RPPA use include biomarker quantification in clinical samples, mapping signaling interactions through phosphorylation events, and assessing molecular drug targets. This method has many advantages over other proteomic techniques such as western blotting, mass spectrometry, or ELISA.

 

The RPPA workflow:

Samples for RPPA are prepared in a similar way to western blot samples using SDS lysis and heat-mediated denaturation. These lysates are then plated into a 384- or 1536-well microtiter plate. Next, a microarrayer prints the samples onto a membrane-coated glass using a solid pin system that can handle the high viscosity of the concentrated samples, creating a “dot” on the membrane. Samples can be run in triplicates, and standard curves are often used. The slides are then blocked and probed with a primary antibody. Immunodetection is performed by an HRP-conjugated secondary antibody; however, a further signal amplification step is often required due to the low protein amount and small dots. The signal is then detected as either brightfield (DAB), a luminescent, or a fluorescent signal. Signal detection and quantification is automated, and the data can then be directly analyzed through software packages. See Figure 1 for an overview of the RPPA workflow.

 

Diagram of simplified RPPA workflow

Fig 1: Simplified workflow of RPPA. Diagram adapted from Akbani et al, 2014 (PMID: 24777629).

 

Advantages of RPPA:
  • The immunochemical signal amplification is greater (via DAB, luminescent, or fluorescent signal) and detection is more sensitive in RPPA, enabling a small amount of protein to be used.
  • Signal readout and quantification is automated, increasing throughput and reducing human error.
  • RPPA offers the possibility for multiplexing when using primary antibodies from different species (e.g., mouse and rabbit).
  • RPPA offers signal uniformity as very large numbers of samples are read at once and can therefore be accurately compared for the same protein.

 

RPPA vs. other proteomic tools:
  • Western blotting is limited in the number of samples that can be compared in a single experiment due to the multi-step protocol including SDS-PAGE and protein transfer to a membrane. Western blot requires 30-50ug of protein lysate, which can be challenging to extract from certain samples. In comparison, RPPA only requires 5ug extracted protein per sample.
  • Mass spectrometry is another powerful proteomic tool and has the advantage of quantifying most of the sample’s proteome at once as well as detecting protein isoforms. It is best used during protein discovery rather than targeted research. However, mass spectrometry is limited to whole proteins and cannot detect post-translational modifications, therefore limiting its application in cell signaling research. RPPA is far more sensitive and cost-effective when investigating a known target. Mass-spec also requires extensive sample preparation, limiting the number of samples that can be processed at once.
  • ELISAs are a sensitive and medium-throughput proteomic technique; however, they are limited to proteins in solution. RPPA can detect proteins from tissue/biopsies or even FFPE samples and therefore is better suited for high throughput clinical use than the other proteomic tools mentioned. ELISAs require pre-determined antibody pairs for detection, whereas RPPA is available to any antibody suited for western blot.

 

Summary Table:

Technique

Advantages

Disadvantages

Western Blot

Protein separation helps ensure the protein detected is the correct one.

Labor intensive

Low throughput

High amount of protein

ELISA

Quantitative

Sensitive

Only for certain samples

Specific antibody pairs required

High amount of protein

Mass spectrometry

Unbiased for de novo protein discovery

Protein isoforms are detected

Thousands of proteins detected at once

Complex sample preparation

Low throughput

Cannot detect PTMs

IHC/IF

Sub-cellular or tissue protein localization

 

Semi-quantitative

Difficult to detect low-concentration targets

RPPA

Low amount of protein (5ug)

High throughput

High sensitivity

PTMs detected

Quantitative

Possibility of multiplexing

Signal uniformity across 1000s of samples.

Special equipment required

High-specificity antibody required for each slide

 

 

The quality of the primary antibodies is at the center of high-quality data from RPPA.

As the protein lysate is not separated by molecular size before antibody probing, the antibody must be tried, tested, and trusted to detect the correct protein prior to large-scale arrays. It is recommended to carry out a western blot with positive and negative controls to confirm the specificity of the antibody for its target.

 

Proteintech antibodies can be used for RPPA!

A recent publication in The Journal of Immunology (Fan et al. 2022; PMID: 36150727) explores the role of metabolic rewiring in human macrophages and lung cancer tissue organoids. Macrophages can display opposing functions depending on their polarization status, and this translates into the cancer context. Tumor-associated macrophages are typically M2-like and tumor-promoting by creating an immunosuppressive environment through T-lymphocyte down-regulation. This is particularly true in Non-Small Cell Lung Carcinoma (NSCLC) where there are areas of T cell exclusion that lead to checkpoint therapy resistance.

The authors used radioactive tracers and Reverse Phase Protein Array (RPPA) to track immunomodulatory metabolic changes over time in healthy and tumor-associated macrophages in response to differential polarization and whole protein glucans (WPGs).

RPPA is a microarray antibody-based technique that allows for high throughput protein quantification. 80% of the primary antibodies used by the authors for RPPA were from Proteintech (42 Proteintech antibodies used in a single publication!). We are honored to help the researchers in the huge undertaking that is mapping macrophage metabolism over time. We are always thrilled to share our customers’ success.

 

Proteintech antibodies used by authors:

1

Caspase 3/p17/p19 Rabbit PolyAb

19677-1-AP

2

CD38 Mouse McAb

60006-1-Ig

3

Citrate synthase Rabbit PolyAb

16131-1-AP

4

CSF1R Rabbit PolyAb

25949-1-AP

5

GM-CSF Rabbit PolyAb

17762-1-AP

6

FBP1 Rabbit PolyAb

12842-1-AP

7

FH Rabbit PolyAb

11375-1-AP

8

GLUD1 Rabbit PolyAb

14299-1-AP

9

GSK3B Rabbit PolyAb

22104-1-AP

10

GYS1 Rabbit PolyAb

10566-1-AP

11

HIF1a Rabbit PolyAb

20960-1-AP

12

HK3 Rabbit PolyAb

13333-1-AP

13

IDH1 Mouse McAb

66197-1-Ig

14

IDH2 Rabbit PolyAb

15932-1-AP

15

IDO1 Rabbit PolyAb

13268-1-AP

16

IFN Gamma Rabbit PolyAb

15365-1-AP

17

IL-1 Beta Rabbit PolyAb

16806-1-AP

18

IL-4 Mouse McAb

66142-1-Ig

19

IL-6 Mouse McAb

66146-1-Ig

20

IL-10 Mouse McAb

60269-1-Ig

21

IL-23A Mouse McAb

66196-1-Ig

22

KGA/GAC Rabbit PolyAb

12855-1-AP

23

MAF Rabbit PolyAb

55013-1-AP

24

MDH1 Rabbit PolyAb

15904-1-AP

25

OGDH Rabbit PolyAb

15212-1-AP

26

Pyruvate Carboxylase Rabbit PolyAb

16588-1-AP

27

PCK1 Rabbit PolyAb

16754-1-AP

28

PCK2 Rabbit PolyAb

14892-1-AP

29

GLUT1 Rabbit PolyAb

21829-1-AP

30

PFKFB3-Specific Rabbit PolyAb

13763-1-AP

31

PGD Rabbit PolyAb

14718-1-AP

32

PKM2-specific Mouse McAb

60268-1-Ig

33

PYGB Rabbit PolyAb

55380-1-AP

34

PYGL Rabbit PolyAb

15851-1-AP

35

QPRT Rabbit PolyAb

25174-1-AP

36

SDHB Rabbit PolyAb

10620-1-AP

37

TGF Beta 1 Rabbit PolyAb

21898-1-AP

38

TNF Alpha Mouse McAb

60291-1-Ig

39

VEGFA Rabbit PolyAb

19003-1-AP

40

G6PD Rabbit PolyAb

25413-1-AP

41

LDHA-Specific Rabbit PolyAb

19987-1-AP

42

PDH E1 Alpha Rabbit PolyAb

18068-1-AP

 

Blog written by Lucie Reboud, 4th year PhD Student at the University of Manchester and Science Marketing Intern for Proteintech.

 

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