Validation on different cell models from different species

The adherent cell lines HeLa (human), NIH-3T3 (mouse), GH1 (rat) and CHO-K1 (hamster) were plated in 96-well culture plates at different cell densities and incubated for 24 hours at 37°C-5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 (1X). The suspension human cell line Jurkat was dispensed under 30 µL in a 96-well half-area plate at different cell densities, incubated for 1h at 37°C-5% CO2, and directly lysed with 10 µL of supplemented lysis buffer #1 (4X). For HTRF detection of GAPDH levels, 4 µL of cell lysate (undiluted or prediluted in the supplemented lysis buffer) were transferred into a low volume white microplate and supplemented with 12 µL of diluent #11 before adding 4 µL of premixed detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

The HTRF GAPDH Housekeeping kit is adapted to adherent and suspension cells from different species. It has been validated on samples from human, mouse, rat and hamster origin, and is also compatible with monkey species. The assay range and sensitivity have been optimized on the most commonly used cell lines and cell densities. For some cell models that express high levels of GAPDH, the lysate must be prediluted in the supplemented lysis buffer 1X before transfer to the detection plate (e.g. 1/4 predilution for NIH-3T3 and CHO-K1 lysates; and 1/5 predilution for Jurkat cell lysates).

 

 

Compatibility with HTRF Phospho-Total protein lysis buffers

GH1 cells were plated at different cell densities in 96-well culture plates, grown for 24 hours at 37°C-5% CO2, and then lysed with HTRF Phospho-Total protein lysis buffer #1, #2, #3 or #4 (supplemented with the blocking reagent). For HTRF detection of GAPDH levels, 4 µL of cell lysate were transferred into a low volume white microplate and supplemented with 12 µL of diluent #11 before adding 4 µL of premixed detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

The assay is compatible with all Phospho-Total protein lysis buffers. The best performance is obtained with lysis buffer #1. However, the three other lysis buffers provide comfortable assay windows (S/N > 8), even on the lowest cell density. These results demonstrate that the GAPDH Housekeeping assay can be performed on the same lysate as the one used for Phospho- and Total protein assays. For more information, please refer to the lysis buffer compatibility table.

 

Control of GAPDH levels in parallel of Phospho- and Total protein assays

Study of ERK inhibition by the MEK inhibitor PD98059 in HeLa cells

HeLa cells were plated in a 96-well culture plate (100,000 cells/well) and incubated for 24 hours before treatment with increasing concentrations of the MEK inhibitor PD98059 for 30 minutes. After cell lysis with 50 µL of supplemented lysis buffer #1, the same lysate was sequentially dispensed into a low volume detection white plate: twice 16 µL for the combined analysis of phospho-ERK and total ERK levels using HTRF Advanced phospho-ERK and Total ERK kits (64AERPET/G/H and 64NRKPET/G/H), and an additional 4 µL (suplemented with 12 µL diluent #11) to control GAPDH levels using the HTRF GAPDH Housekeeping kit. The corresponding kit detection reagents were added under 4 µL and HTRF signals were recorded after an overnight incubation.

Increasing doses of PD98059 lead to increased inhibition of ERK phosphorylation. Since inhibition of ERK phosphorylation occurs without a decrease in total ERK or GAPDH levels, toxicity can be excluded from the list of possible mechanisms of action. Thus, it can be ascertained that PD98059 acts to decrease ERK phosphorylation primarily by targeting MEK, the kinase upstream of ERK.

 

 

 Study of AKT activation by IGF-1 in HEK293 cells

HEK293 cells were plated in a 96-well culture plate (100,000 cells/well) and incubated for 24 hours before treatment with increasing concentrations of human IGF-1 for 10 minutes. After cell lysis with 50 µL of supplemented lysis buffer #1, the same lysate was sequentially dispensed into a low volume detection white plate: twice 16 µL for the combined analysis of phospho-AKT and total AKT levels using HTRF Phospho-AKT (Ser473) and Total AKT kits (64AKSPET/G/H and 64NKTPET/G/H), and an additional 4 µL (suplemented with 12 µL diluent #11) to control GAPDH levels using the HTRF GAPDH Housekeeping kit. The corresponding kit detection reagents were added under 4 µL and HTRF signals were recorded after an overnight incubation.

Cell stimulation with IGF-1 induces an increase in AKT phosphorylation on Ser473 and a down-regulation of AKT expression level, both in a dose-dependent manner with similar EC50 and IC50 values. On the other hand, the level of GAPDH remained unchanged, suggesting that the decrease in total AKT was not caused by an inhibition of global protein synthesis or by experimental issues. This example clearly illustrates the need to analyze the phospho- and total protein of interest, as well as GAPDH, in order to correctly understand the effect of compounds on the protein.

 

 

Correlation with the BCA protein assay

Lysates of Jurkat, CHO-K1, HeLa and NIH-3T3 cell lines plated at different cell densities in 96-well plates were prepared as described in the first section. The level of the housekeeping protein GAPDH was measured in these samples using the HTRF assay. In parallel, the concentration of proteins was determined from the same lysates using the BCA protein assay (QuantiPro™ BCA Assay Kit, Sigma-Aldrich) according to the manufacturer’s instructions. For both methods, lysates were analyzed either undiluted or prediluted in the appropriate buffer, thus ensuring all samples were tested within the respective assay linear ranges. Each graph represents the HTRF GAPDH Signal to Noise (S/N) versus the concentration of proteins determined by BCA. The cell densities used for the correlations are mentioned next to each point.

Using human, mouse or hamster cell lines, the level of GAPDH measured by HTRF is always properly correlated to the concentration of proteins determined by the BCA method (with correlation coefficients r > 0.97). These results demonstrate that GAPDH is an appropriate housekeeping protein suitable for checking the effect of compounds on protein(s) of interest.

 

 

 

 

Validation on tissue samples

Liver tissue samples from three different mice were homogenized with supplemented lysis buffer #3, and one rat liver tissue sample was lysed with supplemented lysis buffer #4. The samples were prepared and analyzed as described in the technical note. The level of the housekeeping protein GAPDH was measured in these samples using the HTRF assay. In parallel, the concentration of proteins was determined from the same lysates using the BCA protein assay (QuantiPro™ BCA Assay Kit, Sigma-Aldrich) according to the manufacturer’s instructions. For both methods, the lysates were analyzed after predilution in the appropriate buffer (dilution factor from 1/50 to 1/200), thus ensuring all samples were tested within the respective assay linear ranges.

The GAPDH Housekeeping assay is compatible with tissue lysates from different species. The assay sensitivity enables a comfortable detection of GAPDH in samples containing less than 0.1 mg/mL of proteins. The level of GAPDH measured by HTRF is always properly correlated to the concentration of proteins determined by the BCA method. These results demonstrate that GAPDH is an appropriate housekeeping protein that can be used to normalize tissue samples in order to study the effect of compounds on animal models.