G-LISA® Activation Assays Technical Guide

Table of Contents

Section I: Introduction

Technical Guides are an accompaniment to the G-LISA kit Protocols and are designed to provide additional information to the end user that may be of use when designing or troubleshooting an activation assay.

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For assistance, contact Technical Support at 303-322-2254 or e-mail [email protected].

Section II: Preparation of Lysates for G-LISA

Recommended lysate amounts per assay

The G-LISA range of activation assays generally requires 6.25-50 µg of lysate per assay. Table 1 summarizes the recommended concentrations of lysate for each small G-protein (GTPase) target. The values in Table 1 are recommended starting points for optimizing a given G-LISA assay. Lysates that are too far above the recommended range may contain a high level of GAPs (GTPase activation proteins) that can inactivate the target GTPase, even in lysis buffer. Final lysate concentrations will vary depending upon experimental factors such as:

A) Total amount of a given GTPase in your cell line or tissue: In general cell lines or tissue that have a high endogenous level of the target GTPase will give a more robust response to an activator.

B) Degree of activation achieved under your experimental conditions: As a general guideline roughly 2-10% of total cellular small G-protein is activated in response to a given stimuli.

Lysis buffer compatibility between assays

Lysis buffer composition is critical to optimal performance of a G-LISA assay. In most assays a standard lysis buffer composition (GL36) has been used (see Table 2). This allows the end user to test various GTPase activities in the same lysate.

For Arf6 analysis one can dilute a GL36 lysate 1:1 with water to achieve the correct assay lysis buffer.

It should be noted that the Cdc42 lysis buffer (GL35) is not compatible with GL36 lysates.

Growth and treatment of tissue culture cells

The health and responsiveness of your cell line is the single most important parameter for the success and reproducibility of GTPase activation assays. Growth conditions vary widely between cell lines, therefore the reader is referred to Tables 3A-H for helpful references regarding GTPase activation in a variety of cell lines. Optimal GTPase activation/inhibition conditions in a given cell line will need to be determined empirically.

Some general guidelines are given below;

1) When possible, untreated samples should have low basal cellular levels of GTPase activity (controlled state). For example, serum starvation often results in low basal levels of GTPase activity and leads to a much greater response to a given activator (see Tables 3A-H).

2) GTPase activation is often very transient, lasting only seconds to minutes. A time courses of activation/inhibition and titration of activating or inhibiting factors are highly recommended when establishing assay conditions.

3) When possible cells should be checked for their responsiveness (responsive state) to a known stimulus (see Tables 3A-H).

4) Poor culturing technique such as sub-culturing of cells that have previously been allowed to become overgrown can result in essentially non-responsive cells. For example, Swiss 3T3 cells grown to >70% confluency should not be used for Rho GTPase activation studies.

5) Cell confluency can affect the cellular response of GTPases to activators/inhibitors. For example RhoA activation generally requires sub confluent cells for a robust response to activators. Confluency restrictions vary widely between different cell lines and different GTPases (see Tables 3A-H).

6) Once an inducible GTPase response has been achieved, it is particularly important to maintain consistent experimental conditions. An Experimental Record Sheet is included in this Guide (Data Analysis section) and is useful for tracking key experimental parameters.

Preparation of lysates from 2D tissue culture cell lines

Activated GTPases are a labile entity and the bound GTP is susceptible to hydrolysis by GAPs during and after cell lysis, resulting in GTPase inactivation. Rapid processing at 4°C is essential for accurate and reproducible results. Detailed instructions for processing lysates from 2D tissue culture cells are given in the G-LISA assay Protocol manuals. The following guidelines are applicable to all G-LISA assays.

1) Wash cells in PBS: Normalization of total cell lysate concentration between samples is required for G-LISA assays. Washing cells in PBS allows removal of protein species from tissue culture media which could interfere with accurate lysate concentration determination.

2) Complete removal of PBS after washing: It is important to maintain the composition of the cell lysis buffer between samples, therefore complete removal of PBS, preferably with an aspirator, should be performed prior to cell lysis.

3) Use cold reagents for processing cells: The PBS used for washing cells and cell lysis buffer should be at 4°C. The cold buffers will help minimize hydrolysis of GTPases during processing. It should be noted, in some cell lines (e.g. 3T3 cells) Cdc42 activation appears to be affected by the temperature of the wash solution. In such cases room temperature PBS may be used. The need to use room temperature PBS should be determined empirically. In all cases, cold lysis buffer supplemented with protease inhibitors should be used.

4) It is recommended to process a “Test plate” prior to the experimental plates. This is used to determine the volume of lysis buffer required to achieve the lysate concentrations recommended in Table 1.

5) Work quickly to process lysates: GTPase proteins are labile entities and will hydrolyze over time. It is recommended to take no more than 15 minutes to process a sample and to process samples sequentially.

6) Snap freeze aliquots of lysate in liquid nitrogen: It is often impractical to process fresh lysates and use them immediately in an activation assay. In particular, if time-points are being taken or multiple samples are being processed, parallel processing is impractical and unnecessary. We highly recommend sequential preparation of samples followed by protein concentration determination, aliquoting and snap freezing aliquots in liquid nitrogen for later analysis. Lysates are stable for several months at -70°C.

7) Keep lysates at 4°C during processing: Lysates should be embedded in ice during processing. This helps to minimize changes in signal over time.

8) Thaw lysates quickly and DO NOT re-freeze: Thawing of cell lysates prior to use in the G-LISA^® assay should be in a room temperature water bath, followed by rapid transfer to ice and immediate use in the assay. Transfer to ice while 1/3 of lysate remains frozen as remainder will thaw quickly on ice. Discard any unused lysate.

Preparation of lysates from 3D tissue culture cell lines

Cells grown in Matrigel or collagen gels can be assayed using the G-LISA format. The low amounts of cells in 3D cultures makes the conventional pull-down assay very difficult, variable and expensive.

Keely et al., 2007 (Methods in Enzymology, v426, p27) and Petroll et al., 2008 (J Cell Physiol, v217, p162) give in-depth descriptions using G-LISA technology and 3-D cultures. An additional modification we suggest is to increase the ratio of extract/binding buffer from the recommended 1:1 (v/v) to 1:3

Preparation of lysates from tissue samples

Tissue lysates can be used in G-LISA assays. Recommendations regarding tissue lysates are given below.

1) Ras superfamily GTPases are labile proteins that will hydrolyze bound GTP during sample handling. Tissues should therefore be processed quickly (2 min) and at 4°C. Tissues should be processed immediately using 4°C buffers or cut into small chunks (1 mm diameter), snap frozen in liquid nitrogen, and stored at -70°C for later processing.

2) Tissues can be homogenized using a micro-pestel on ice. Homogenates should be clarified by a 1 minute centrifugation at 4°C. Lysates can be used immediately in an activation assay or snap frozen in “experiment-sized” volumes. We strongly recommend snap-freezing lysates in liquid nitrogen.

3) When possible, tissues should be extracted in the respective G-LISA’s cell lysis buffer as this is the recommended buffer for the respective G-LISA assay (see Table 1).

4) It is recommended that lysis buffer be supplemented with protease inhibitors and phosphatase inhibitors. Recommended inhibitors include: Cytoskeleton protease inhibitor cocktail (Cat# PIC02), sodium fluoride (50 mM final), sodium pyrophosphate (20 mM final), p-Nitrophenyl phosphate (1 mM final), and microcystin LR (1 µM final).

Equalization of Cell Lysate concentrations

Equal protein concentration in all samples is a prerequisite for accurate comparison between samples in GTPase activation assays. Cell extracts should be equalized with ice-cold Lysis Buffer containing protease inhibitor cocktail to give identical protein concentrations. See Table 1 for recommended lysate protein concentrations for each G-LISA. Equalization is not necessary if the variation between samples is less than 10%.

The Precision Red Advanced Protein Assay Reagent, supplied in all G-LISA kits, is a simple one step procedure that results in a red to purple/blue color change characterized by an increase in absorbance at 600 nm.

The assay exhibits low variance in readings between different proteins of the same concentration and high reproducibility of the colorimetric response. The assay can also be performed in approximately 1-2 minutes. These properties are particularly valuable when applied to the labile lysates required for activation assays.

Quick Protein Concentration Method for 1 ml Cuvette (recommended)

Add 20 µl of each lysate or Lysis Buffer into disposable 1 ml cuvettes.
Add 1 ml of Precision Red^TM Advanced Protein Assay Reagent to each cuvette.
Incubate for 1 min at room temperature.
Blank spectrophotometer with 1 ml of Precision Red plus 20 µl of Lysis Buffer at 600 nm.
Read absorbance of lysate samples.
Multiply the absorbance by 5 to obtain the protein concentration in mg/ml

Example Calculation

Assume a 20 µl sample of cell lysate added to 1 ml of Precision Red reagent gives an absorbance reading of 0.1.

Where c = protein concentration (mg/ml), A = absorbance reading, l = pathlength (cm), ε = extinction coefficient ([mg/ml]^-1 cm^-1) and the multiplier of 50 is the dilution factor for the lysate in Precision Red reagent (20 µl lysate in 1 ml Precision Red reagent). 

Thus, for a 20 µl sample in 1 ml of Precision Red, the equation becomes C = A x 5

For a 10 µl sample in 1 ml Precision Red, the equation becomes C = A x 10

Quick Protein Concentration Method for 96 Well Plate

• Add 10 µl of each lysate or Lysis Buffer into the well of a 96 well plate.
• Blank spectrophotometer with 290 µl of Precision Red plus 10 µl of Lysis Buffer at 600 nm.
• Add 290 µl of Precision Red^TM Advanced Protein Assay Reagent to each well.
• Incubate for 1 min at room temperature.
• Read absorbance of lysate samples.
• Multiply the absorbance by 3.75 to obtain the protein concentration in mg/ml

96 Well Plate Method

The linear range of this assay is 0.05 - 0.4 and is recommended when lysates are below the linear range of the 1 ml cuvette method. The pathlength for 96 well plate readings is 0.8 cm, hence the equation is modified as shown in the example below:

Example Calculation for 96 Well Plate Measurement

Assume a 10 µl sample of cell lysate added to 290 µl of Precision Red reagent gives an absorbance reading of 0.1

Where c = protein concentration (mg/ml), A = absorbance reading, l = pathlength (cm), ε = extinction coefficient ([mg/ml]^-1 cm^-1) and the multiplier of 30 is the dilution factor for the lysate in Precision Red reagent (10 µl lysate in 290 µl Precision Red reagent).

Thus, for a 10 µl sample in 290 µl Precision Red, the equation becomes C = A x 3.75

For a 5 µl sample in 295 µl Precision Red, the equation becomes C = A x 7.5

NOTE: The protein concentrations generated by the multipliers stated in the method will result in approximate lysate concentrations. Data will be highly reproducible from lysate to lysate and will generate excellent values for relative concentrations of a series of lysates. It should be noted for activation assays, the relative protein concentration between experimental extracts is far more important than the absolute protein quantitation. However, if desired, one can create a standard curve using BSA or IgG protein standards for each experiment. The standard curve should be performed prior to lysate preparations due to the labile nature of the lysates.

Section III: Assay Technical Tips

Plate shaker recommendations

It is recommended to use an orbital plate shaker at 400 rpm. As a back-up you can use a 200 rpm orbital shaking culture incubator or a normal orbital rotating platform set to 200 rpm. Signals may be lower with the 200 rpm option. Rocking or tilting plate shakers will not be sufficient for this assay.Below are some examples of acceptable plate shakers:

Model # 4625, Titer Plate Shaker, Lab-Line Instruments, Barnstead Intl.

Model # RF7854, Digital Microplate Shaker, ML Market Lab, researchml.com

Model # RF7855, Incubating Microplate Shaker, ML Market Lab, researchml.com

Vortex of samples after binding buffer addition

Binding buffer is a viscous solution and requires thorough mixing after addition to cell lysates. The best way to do this is a brief 3-5 second vortex on high. This step will greatly reduce variability between duplicate samples and will increase overall assay accuracy.

Spectrophotometer / Luminometer settings

Spectrophotometer: The majority of the work in the design of the colorimetric G-LISA assays has been based on the Molecular Devices SpectraMax 250. The spectrophotometer settings are given in Table 4 as a reference.

Luminometer: Luminometers vary widely in their sensitivity and absolute readings. It is therefore recommended to run a G-LISA assay with blank and positive control to determine that you are in the linear range of the assay. When in the linear range of the assay the positive control should read 4-10 fold higher than the blank wells. Table 5 gives guidelines for luminometer settings;

Section IV: Data Analysis

Data Analysis: Excel format

6. Enter your replicate data into rep1, rep2 etc. It doesn’t matter if you only have duplicates because the program will ignore any sectors that do not contain data. The program will calculate the Mean and Standard Deviation of your replicates.

7. When the data has been entered select the Sample, Mean and Standard Deviation data sectors by the click and drag method. Then select the chart making function, in Excel this looks like a clickable square with a mini-bar chart inside. This will guide you through the chart making process with the data you have selected. Choose “column chart” initially, designate the Mean numbers for input values. The Standard Deviation column for the y-axis error bars needs to be designated after the Mean numbers chart is made. This is achieved by double clicking on the graph bars, and selecting the “Y-axis error” tab, then entering the location of the Standard Deviation data by clicking the “Custom” option and selecting the area in the worksheet. E-mail [email protected] or visit cytoskeleton.supremeclients.com to download a free Excel Template.

8. An example of a typical Excel layout and data plot is shown in Figure 1.

Download Experiment Record Sheet

Download Plate Record Template

Technical Assistance: call either 303-322-2254 or e-mail [email protected]