Protocol for simultaneous detection of dual subcellular localized dengue virus protease by co-transfection

Summary

It is difficult to track virus-coded proteins simultaneously if they localize to multiple subcellular organelles. Here, we present a protocol for the simultaneous detection of dual subcellular localized dengue virus protease by co-transfection. We describe steps for cell seeding, co-transfection with mitochondria targeted red fluorescent protein, cell fixation, permeabilization, and staining of transfected cells with Hoechst stain. Further, we describe how to generate fluorescent intensity profiles using the NIS-Elements software. We then detail procedures for subcellular fractionation followed by western blotting.

For complete details on the use and execution of this protocol, please refer to Gandhi et al.¹

Graphical abstract

Highlights

• •Co-transfection and staining with Hoechst for dengue virus protease localization
• •Simultaneous detection of protease in nucleus and mitochondria by confocal microscopy
• •Confirm observations via fluorescence intensity profiles from the NIS-Elements software
• •Western blotting analysis of subcellular fractions

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

It is difficult to track virus-coded proteins simultaneously if they localize to multiple subcellular organelles. Here, we present a protocol for the simultaneous detection of dual subcellular localized dengue virus protease by co-transfection. We describe steps for cell seeding, co-transfection with mitochondria targeted red fluorescent protein, cell fixation, permeabilization, and staining of transfected cells with Hoechst stain. Further, we describe how to generate fluorescent intensity profiles using the NIS-Elements software. We then detail procedures for subcellular fractionation followed by western blotting.

Before you begin

Prepare the plasmids using Midi preps for this protocol in order to acquire sufficient quantity and quality required for the transient transfections. Autoclave and expose the coverslips to ultraviolet (UV) light before use. Although HEK cells are used in the present experiment, any other adherent cell lines could be used in this protocol based on their doubling time, required buffer conditions and incubation times. This protocol is described with the Green fluorescent (GFP) and Red fluorescent (MitoRFP) fusion proteins. But, fluorescent tagged antibodies or the Mito tracker dye also can be used for localization analysis.^2,3

Culturing and maintenance of cells

Timing: 45 min each day, 4–5 days

In this section, we have described the routine cell culture practice for maintaining the cell lines.

• 1.Thaw 2.0 mL cryovial of fresh HEK (∼1 × 10⁴) cells stored in liquid nitrogen or at ‒80°C.
• 2.Suspend the cells in 1 mL of serum free Dulbecco’s Modified Eagle’s Medium (DMEM). Centrifuge the suspension at 500 × g for 5 min.
• 3.Resuspend the cells in 1 mL of fresh DMEM containing heat-inactivated 10% (v/v) Fetal Bovine Serum (FBS) and 1% antibiotics (penicillin and streptomycin).
• 4.
  Take 100 μL of cell suspension and 100 μL of trypan blue dye.

  • a.
    Place the coverslip on the hemocytometer.
  • b.
    Add 10 μL of trypan blue cell suspension on the coverslip gently with the pipette. Allow the cells to spread evenly on the counting chamber.

    • i.
      Count the number of viable cells in 4 big squares of hemocytometer.
    • ii.
      Calculate the cell number using the following formula.
      No of cells/mL = (No. of cells in 4 big squares/4) × 10⁴ × dilution factor∗
      Dilution factor∗ = 2 (100 μL cell suspension: 100 μL trypan blue).
      10⁴ = Conversion factor to 1 mL.
      For example: No of cells in 4 big squares: 200.
      No of cells/mL = (200/4) × 10⁴ × 2 = 1,000000 = 1 × 10⁶ cells/mL.
      Since 1 × 10⁶ cells in 1000 μL, it is 7 × 10⁵ cells in 700 μL of DMEM.
• 5.Mix 700 μL (∼7 × 10⁵ cells) of the above cell suspension with 2.3 mL of fresh complete DMEM and seed in T-25 flask.
• 6.Incubate at 37°C in a humidified incubator for 12–16 h with 5% CO₂.
• 7.Observe the cell’s adherence on the next day under an inverted bright field microscope and replace the medium with 3 mL of fresh complete DMEM.
• 8.Split and culture the cells for 4–5 times in order to get the active cells with confluency up to 70%–80%.

CRITICAL: To attain a good transfection efficiency, cells must split 2–3 times before transfection experiments.

Day prior to transfection

Timing: 1 h

This section follows up with the above protocol for maintaining, cell counting and seed the cells before the transfection experiments.

Cell counting and seeding

• 9.
  Add 1 mL of Trypsin-EDTA solution (5X) and incubate the cells at 37°C in a CO₂ incubator for 3 min for trypsinization.

  • a.
    Add 500 μL of DMEM containing 10% FBS to inactivate trypsin.
  • b.
    Harvest the cells by centrifuging at 500 × g for 3 min.
• 10.
  Add 500 μL of 1X PBS/DMEM (without serum) in 1:1 ratio to the pelleted cells.

  • a.
    Centrifuge at 500 × g for 3 min.
  • b.
    Repeat the above washing.
  • c.
    Resuspend the cells in 1 mL of complete DMEM.
• 11.
  Place the clean and UV-treated coverslips in 12-well plates. (See the troubleshooting 1).

  • a.
    Add trypan blue dye to the cells in a hemocytometer and count the cells.

    • i.
      Seed the cells in the above UV treated 12 well plates (8 × 10ˆ4 to 1 × 10ˆ5 cells per well).
    • ii.
      Allow the cells to grow for ∼16 h at 37°C in a humidified CO₂ incubator to reach up to 70%–80% confluency.
  • b.
    For subcellular fractionation, seed 1 × 10ˆ6 cells in 60 mm dishes.

CRITICAL: Since the HEK cells are semi-adherent and get removed, cell counting is critical in avoiding over confluency. 60%–70% confluency would be sufficient for the localization studies.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
GFP (D5.1) rabbit mAB (monoclonal antibody) (1:1,000)	Cell Signaling Technology, USA	Cat# 2956, RRID: AB_1196615
Actin mouse monoclonal antibody (1:2,500)	Santa Cruz Biotechnology	Cat# sc8432
Rabbit anti-mouse IgG HRP-conjugated antibody (1:10,000)	GeNei, India	Cat# 1140580011730, RRID: N/A
Mouse anti-rabbit IgG HRP-conjugated antibody (1:10,000)	Santa Cruz Biotechnology	Cat# sc-2357, RRID: AB_628497
NS2BNS3pro antibody (1:1,000)	Raised in house	N/A
Chemicals, peptides, and recombinant proteins
NaCl	Merck	Cat# 1.93206.0521
Tris base	HiMedia	Cat# TC072
NaH2PO4	Merck	Cat# MB024
KH2PO4	HiMedia	Cat# PCT0009
KCl	HiMedia	Cat# 7447-40-7
MgCl2	MP Biomedicals	Cat# 209844
EDTA	SR Life Sciences	Cat# 50027
HEPES buffer	HiMedia	Cat# RM380
Glycerol	HiMedia	Cat# TC503
Trypan blue	Sigma	Cat# T6146
DMEM	Gibco	Cat# 11995-065
FBS	Gibco	Cat# 10270106
Anti-anti (antibiotic)	Gibco	Cat# 15240062
Trypsin-EDTA (5X) solution	HiMedia	Cat# TCL007
Protease inhibitor cocktail (ProteaseArrest)-100X	G-Biosciences	Cat# 786-331
Bradford reagent	Bio-Rad	Cat# 5000201
Immobilon-P PVDF membrane	Merck Millipore	Cat# IPVH00010
Paraformaldehyde	Sigma	Cat# P6148
Triton X-100	HiMedia	Cat# TC286
RIPA buffer	Sigma-Aldrich	Cat# R0278
Hoechst stain 33342	Molecular Probes	Cat# H21492
femtoLUCENT PLUS-HRP	G-Biosciences	Cat# 786-003
Lipofectamine reagent 2000	Invitrogen	Cat# 11668-027
Experimental models: Cell lines
Human embryonic kidney 293 cells (HEK293)	National Centre for Cell Science (NCCS), Pune, India	N/A
Recombinant DNA
pEGFP-N1 NS2BNS3 helicase	Gandhi et al.1	https://doi.org/10.1016/j.isci.2023.107024
pEGFP-N1 NS3pro helicase	Gandikota et al.6	https://doi.org/10.1128/JVI.01178-20
MitoRFP vector	Gandikota et al.6	https://doi.org/10.1128/JVI.01178-20
pEGFP-N1 vector	Gandikota et al.6	https://doi.org/10.1128/JVI.01178-20
Software and algorithms
NIS-Elements AR software	Nikon Microscopes	N/A
Other
Cell culture 12-well plates	Tarsons	Cat# 980040
60 mm dishes	Nunc, Thermo Fisher Scientific	Cat# 150462
T-25 flasks	Nunc, Thermo Fisher Scientific	Cat# 156367
CO2 incubator	Eppendorf Galaxy	Cat# Galaxy 48R
Hemocytometer	Neubauer blood counting chamber	N/A
Laser scanning confocal microscope	Carl Zeiss	LSM170
‒80°C deep freezer	Eppendorf	Cat# f570
SDS-PAGE unit (Mini-PROTEAN tetra cell)	Bio-Rad	Cat# 1658038
Microcentrifuge	Eppendorf	Cat# 5424R
Electroblot transfer unit	Bionova	N/A
ChemiDoc image analyzer	Bio-Rad	Cat# 12003028
Inverted bright-field microscope	Lawrence & Mayo	Cat# TC5400

Materials and equipment

Phosphate Buffer Saline (PBS) 10X

Reagent	Stock concentrations (10X)	Amount in grams for 1 L
NaCl	1.37 M	80
NaH2PO4	100 mM	17.8
KH2PO4	18 mM	2.4
KCl	27 mM	2.0
H2O	-	Up to 1 L

Note: Adjust pH to 7.4

Stock can be stored at 25°C up to 1 month.

Note: 500 mL of 1X PBS can be prepared from 10X stock: Add 50 mL of 10X PBS to 450 mL (can be stored at 25°C for a week).

0.25% Triton X-100 buffer

Reagent	Final concentration	Amount
Triton X-100	0.25%	25 μL
1X PBS	-	10 mL

Stock can be stored at 25°C for 1 week and at 4°C up to 1 month.

Hoechst 33342 stain solution

Reagent	Stock concentrations	Amount
Hoechst 33342 stain	10 mg/mL	10 mg
H2O	N/A	1 mL

Working solution can be prepared by dissolving the stock in 1X PBS (1:1,000).

Stock solution can be stored at 4°C up to 8–12 months and protected from light.

Hypotonic Buffer

Reagent	Stock concentrations	Final concentrations	Amount
HEPES (pH 7.9)	1 M	20 mM	1 mL
KCl	1 M	10 mM	500 μL
EDTA	100 mM	1 mM	500 μL
Glycerol	100%	10%	5 mL
Triton X-100	100%	0.5%	250 μL
H2O	N/A	-	40.50 mL

EDTA can be stored at ‒20°C up to 1–3 months.

Stocks can be stored at 4°C up to 1 month.

Hypertonic Buffer

Reagent	Stock concentrations	Final concentrations	Amount
HEPES (pH 7.9)	1 M	20 mM	1 mL
KCl	1 M	10 mM	500 μL
NaCl	1 M	240 mM	12 mL
EDTA	100 mM	1 mM	500 μL
Glycerol	100%	20%	10 mL
Triton X-100	100%	1%	0.5 mL
H2O	N/A	-	25.50 mL

Stocks can be stored at 4°C up to 1 month.

1X PBS-T buffer

Reagent	Amount (for 1 L)
10X PBS	100 mL
Tween-20	1 mL
H2O	to 1 L

Stocks can be stored at 25°C up to 1 week.

Blocking buffer

Reagent	Amount for 100 mL
BSA or Skimmed milk (5%)	5 g
1X PBS-T	100 mL

Always prepare fresh.

10X Transfer Buffer

Reagent	Concentration	Amount for 1 L
Tris	25 mM	30.2 g
Glycine	192 mM	144 g
H2O	-	Up to 1 L

Stock can be stored at 4°C for up to 3 months.

Note: 1X Transfer buffer can be prepared from 10X stock: Dissolve 100 mL of 10X transfer buffer in 200 mL of methanol and make up the volume with water up to 1 L (store at 4°C and can be re-used for 2 weeks).

RIPA buffer (1X): Ready to use buffer containing 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris. pH 8.0.

Can be stored at 4°C up to 3–4 months.

See key resources table.

Reagent	Stock concentrations	Amount for 1 mL
Protease Inhibitor cocktail	100X	100 μL
H2O	-	900 μL

Protease inhibitor cocktail (10X)

Note: Aliquots of 10X Stock can be stored at ‒20°C up to 3–4 months or at 4°C for 1 month. Do not repeat freeze thaw.

10X stock can be directly used to make 1X (final concentration) according to volume of RIPA buffer required for extraction procedure.

Ponceau S solution

Reagent	Final concentrations	Amount for 500 mL
Ponceau S	0.1%	0.5 g
Glacial Acetic Acid	5%	25 mL
H2O	-	475 mL

Store the solution at 25°C up to 1 month.

Precaution: Glacial acetic acid is corrosive acid. Handle with care- use fume hood and gloves.

Primary antibody dilution and incubation conditions

Primary antibodies	Dilution	Solution	Incubation condition/times
NS2BNS3pro	1:1,000	1X PBST or 2% skimmed milk	4°C for 12–14 h
Anti-GFP tag	1:1,000	1X PBST	25°C for 2 h or 4°C for 12 h
Anti-Actin	1:2,500	1X PBST	4°C for 12 h

Antibody solutions can be stored at ‒20°C for long term use or as directed by the manufacturer’s instructions.

https://www.cellsignal.com/products/primary-antibodies/gfp-d5-1-rabbit-mab/2956.

Secondary antibody dilution and incubation conditions

Secondary antibodies	Dilution	Solution	Incubation condition/times
Anti-Rabbit IgG HRP- conjugated antibody	1:10,000 or 1:20,000	1X PBST or 2% skimmed milk	25°C for 2 h
Anti-mouse IgG HRP-Conjugated antibody	1:10,000 or 1: 20,000	1X PBST	25°C for 2 h

Antibody solutions can be stored at ‒20°C for long term use or as directed by the manufacturer’s instruction

https://www.scbt.com/p/mouse-anti-rabbit-igg-hrp.

https://geneilabs.com/product/rabbit-anti-mouse-igg-hrp-1-ml/.

Step-by-step method details

Co-transfection

Timing: 40 min

This method is described for co-transfecting two recombinant plasmids.

• 1.Observe the cells (HEK) seeded in 12 well plates and the 60 mm dishes under the microscope.

Note: The required confluency is 70%–80%.

• 2.
  Mix 400–600 ng of vector/recombinant vectors (pEGFP-N1 Vector, pEGFP N1-NS2BNS3 and pEGFP-N1-NS3pro helicase) with 400–800 ng of Mito RFP.

  • a.
    Dilute the above mix with serum free DMEM/Opti-MEM (Mix-1).
  • b.
    Similarly, mix Lipofectamine 2000 with serum free DMEM or Opti-MEM (Mix-2).
  • c.
    Incubate both mixes separately at 25°C for 5 min.
  • d.
    The above plasmids were used to analyze the expressed protein of interest which is fused with GFP tags (See the troubleshooting 2 & 3).

Note: The required volume of Lipofectamine to prepare Mix 2 is according to the concentration of plasmid used. In this protocol, 1 μg:2 μL was used.

• 3.Add Mix-1 to Mix-2 and incubate again for 20 min and then add to the cells.
• 4.Allow the cells to grow for 4–6 h at 37°C in a CO₂ incubator.

Note: Transfect the above vector/recombinant vectors (pEGFP-N1, pEGFP-N1 NS2BNS3 and pEGFP-N1 NS3pro helicase) into the cells of 60 mm dishes by following the same transfection method but without MitoRFP for subcellular fractionation analysis.

• 5.Replace the serum free medium with fresh complete DMEM after 5 h, and allow the cells to grow for 48 h for the GFP/MitoRFP expression analysis (See the troubleshooting 3).

Permeabilization and fixation

Timing: 45 min

This method is described for slide preparation for confocal microscopy.

• 6.
  Wash the cells by adding 500 μL of 1X PBS.

  • a.
    Fix the cells by using 500 μL of 4% paraformaldehyde (or 10% Formalin).
  • b.
    Incubate for 10–20 min at 25°C.⁵
• 7.
  Permeabilize the cells with 0.25% Triton X- 100 buffer.

  • a.
    Incubate for 3–5 min at 25°C.
  • b.
    Wash the cells with 500 μL of 1X PBS 2–3 times until Triton X-100 buffer was completely removed.
• 8.Stain the cells with 200 μL of Hoechst stain (for nuclear staining) and incubate in dark for 3 min at 25°C.
• 9.Wash the cells with 1X PBS; remove excess PBS by gently tapping the coverslips onto the tissue paper. Air dry the coverslips and add a drop of glycerol for mounting.
• 10.Analyze the transfected cells using a laser scanning confocal microscope (Carl Zeiss, LSM710) at 20X and 60X magnifications using the fluorescence filters for green (GFP), red (MitoRFP), and blue (Hoechst stain) colors.
• 11.The captured images were merged in order to observe the co-localizations.

Note: In this step, the permeabilization step can be omitted as the fluorescence expressed by the GFP and MitoRFP was analyzed but not with the specific antibodies. The permeabilization step is crucial for the indirect immunostaining method.

Subcellular fractionation

Timing: 45 min (for step 12)

Timing: 30 min (for step 13)

Timing: 45 min (for step 14)

Timing: 2 h

The protocol for subcellular fractionation was followed as per the earlier described methods^4,6,7,8 but with minor modifications. This section describes the detailed fractionation steps for whole cell lysate, cytoplasmic and nuclear fractionations. Perform all the steps at 4°C. Buffers should be sufficient and must be pre-cooled while conducting the experiments.

In order to continue the steps 1–4 under co-transfection experiment, harvest the cells after 48 h by scraping and centrifugation at 800 × g for 5 min. Wash the above obtained cell pellets with 1 mL of 1X PBS by centrifugation at 800 × g for 5 min. Use these cell pellets for the preparation of whole cell lysate/cytoplasmic/nuclear extracts.

• 12.
  Preparation of whole cell lysate.

  • a.
    Resuspend the above cell pellet in 100–120 μL of Radio Immunoprecipitation Assay buffer (RIPA buffer).

    • i.
      Add 10 μL of protease inhibitor cocktail (10X) to 100 μL of lysate and incubate on ice for 30 min.

      Note: Add protease and phosphatase inhibitors to avoid proteolysis.

    • ii.
      Vortex the lysate 2–3 times with an interval of 15 min in order to suspend the pellet completely.
  • b.
    Centrifuge the above suspension at 20,000 × g for 15 min and collect the supernatant as the whole cell lysate.
• 13.
  Preparation of cytoplasmic /mitochondrial fraction.

  • a.
    Wash the cell pellets with 1 mL of ice-cold 1X PBS and incubate on ice for 5 min.

    • i.
      Add 100 μL of hypotonic buffer, homogenize gently with micropipette.
    • ii.
      Incubate on ice for 10 min.
  • b.
    Centrifuge the lysate at 2000–3000 × g for 10 min at 4°C; collect the supernatant as cytoplasmic fraction.
  • c.
    Process the pellet further to obtain the nuclear extract.

CRITICAL: Mild homogenization is crucial for obtaining pure cytoplasmic fractions. The speed of centrifugation varies for each cell line used. For HEK, we have used speed up to 3000 × g.

• 14.
  Preparation of Nuclear Extract.

  • a.
    Wash the nuclear pellet obtained in the above fractionation twice with 1 mL of 1X PBS.

    • i.
      Resuspend in 100 μL of 1X RIPA buffer. ii. Incubate the suspension for 30 min on ice with vortexing at 5 min intervals.
  • b.
    Centrifuge at 20,000 × g for 15 min and collect the supernatant as a nuclear fraction.

Optional: For nuclear fraction, hypertonic buffer can be used. Here we described the extraction with RIPA buffer as it is rapid (30 minutes), easy and efficient for solubilizing nuclear proteins.

Pause points: The fractionated lysates can be stored directly in ‒80°C, if not used immediately or make aliquots of each fraction in small 0.5 mL tubes to avoid repeated free thaw.

Western blotting

Timing: ∼9 h

In this section, we have described the protocol of immunoblotting for the above fractionated lysates.

Quantify the above obtained whole cell lysate, nuclear and cytoplasmic fractions using Bradford reagent and subject to western blotting or store at ‒80°C until further use.

• 15.Resolve the total proteins (60 μg of each) of whole cell lysate, cytoplasmic and nuclear extracts on 10% SDS-PAGE.
• 16.Transfer the resolved proteins onto polyvinylidene fluoride (PVDF) membrane at 80 V current for 2–3 h (or 12 h at 50 V) using western blot transfer unit in 1X transfer buffer.
• 17.Stain the membrane with Ponceau S and record the images. Wash the membranes with 1X PBST until the Ponceau was removed completely.

Note: Ponceau stain must be removed as it may hinder the blocking step.

• 18.Block the membrane using 5%–7% blocking buffer for 2 h at 25°C with gentle rocking.

Note: Blocking step must be optimized for each antibody. (See the troubleshooting 4).

• 19.Dilute the primary antibodies in 1X PBST or 2% blocking buffer.
• 20.Rinse the membrane with 1X PBST and incubate in primary antibody solution for 2 h at 25°C or 12–14 h at 4°C under gentle rocking. (See the troubleshooting 5).

Note: Incubation time and dilution of antibodies must be optimized as per manufacturer’s instructions.

• 21.Wash the membrane three times with 1X PBST each for 10 min on a rocker.
• 22.Incubate the membrane in secondary antibody solution for 2 h at 25°C.
• 23.Wash the membrane three times with 1X PBST each for 10 min on a rocker.
• 24.Develop the membrane with western blot developing solution (femtoLUCENT PLUS-HRP, G-Biosciences) and record the images using ChemiDoc Image System (Bio-Rad).

Expected outcomes

Tracking of extracellular proteins localized in different subcellular compartments is critical in analyzing cell homeostasis. Electron microscopy with gold particles is being used particularly for the nucleus,^9,10 but the limitations are the required expertise and the high cost. Use of radioactivity is another approach for mitochondria¹¹ but the health hazards and its availability restrict these protocols. Fluorescently labeled molecules are being used in this direction and the protocols based on these molecules are simple, economical with wider applications. The present protocol describes the co-transfection of dengue virus NS2BNS3 or NS3 along with MitoRFP. The transfected cells were further stained with Hoechst and observed under a confocal microscope. The NS2BNS3 (GFP) or NS3 (GFP) localized to mitochondria was detected due to the co-localized MitoRFP (Red) and also simultaneously could be detected if the same protein localized to the nucleus due to the Hoechst (blue) staining (Figure 1A, viii & xii). The above data was confirmed by overlapping green (GFP) and red (MitoRFP) peaks in case of NS3 suggesting its localization to mitochondria (Figure 1B, iii). The high GFP expression peaks at the blue peak region suggest the NS3 localization in the nucleus also (Figure 1C, iii). Further, cells of the same experiment were subjected to subcellular fractionation followed by western blotting. This step allows the confirmation of the above confocal data. We expect that this protocol can be used to screen the anti-dengue virus protease molecules as it is simple, easy to perform and interpret the outcome.

Figure 1.

Simultaneous detection of NS3pro-helicase in nucleus and mitochondria

(A) GFP and blue/red fluorescence images captured by confocal microscope at 60X (scale bar = 20 μm). i-iv (vector); v-viii (NS2BNS3); ix-xii (NS3).

(B) Graphical representation of green and red in the whole cell, i-vector; ii-NS2BNS3 and iii-NS3.

(C) Graphical representation of green and blue in the nuclear region, i-vector; ii-NS2BNS3 and iii-NS3.

(D) NS2BNS3 and NS3 subcellular fractions of transfected HEK cell lysates probed with NS2BNS3pro antibody. NS2BNS3 (90 kDa) is detected in nucleus (D i), whereas NS3 (75 kDa) is present in both cytoplasmic (mitochondria) and nuclear fractions (D ii).

Quantification and statistical analysis

Data analysis

Subcellular localization of dengue virus protease forms i.e., NS2BNS3 and NS3 were analyzed in this protocol. Nuclear localization signal (NLS) and mitochondrial targeting sequences (MTS) are present in NS3 but only NLS is present in NS2BNS3.^1,6 Separate filters were used to visualize the GFP & blue (nucleus) (Figure 1A ix & xi) and GFP & red (mitochondria) (Figure 1A x & xi) fluorescences in transfected cells. In the merged images, GFP of NS2BNS3 is localized only to the nucleus (blue) and no GFP is observed in mitochondria (red) suggesting no localization of NS2BNS3 to the mitochondria [Figure 1A (viii)]. This observation was further supported by the graphical analysis in which the red peaks of mitoRFP are completely separated from the green peaks [Figure 1B (ii)]. A high intensity peak of GFP (green peak) in the nuclear region (blue) confirms the localization of NS2BNS3 into the nucleus [Figure 1C (ii)]. But, in the case of NS3, GFP expression is present in nucleus (blue) as well as in mitochondria (red) suggesting the NS3 localization in both nucleus and mitochondria [Figure 1A (xii)]. This observation was further supported by the graphical intensity profiles that the green peak (GFP) is merged with red peak (mitoRFP) [Figure 1B (iii)] and a high GFP intensity in blue peak region (nucleus) [Figure 1C (iii)]. Figure 1A (i-iv); B (i) and C (i) are vector controls.

Using the cell lysates of the above experiments, the western blotting analysis was carried (anti-NS2BNS3pro antibody) to verify the observations. The results were found to be consistent with the above localization protocol suggesting that NS2BNS3 (92 kDa) localized only to the nucleus (Figure 1D i) whereas the NS3pro-helicase (75 kDa) localized to both cytoplasm (mitochondria) and nucleus (Figure 1D ii). The absence of actin (Figure 1D iii) and less GFP (Figure 1D iv) in nuclear fraction support the fractionations.

Note:Figure 1B (i-iii): GFP, MitoRFP and Hoechst stain fluorescences were measured in whole cells and the peaks of GFP and MitoRFP were considered.

Figure 1C (i-iii): GFP and Hoechst stain fluorescences were measured only in nucleus and the corresponding peaks were analyzed.

Image acquisition and quantification of the fluorescence intensities in the transfected cells

Prepare the localization intensity profiles using NIS-Elements AR software program and generate fluorescence intensity graphs. Select the filters GFP, MitoRFP and Hoechst from the software as channel 1- Green, Channel 2- RED, Channel 3- Blue. Select the Region of Interest (ROI) from the confocal image to measure the intensity signal (Figure 2 i). Select the mean intensity and areas for the ROI. This gives the raw data for the intensity of each individual channel. With the obtained raw data, generate the automated fluorescence intensity graph profiles with green, red and blue peak overlapping and nonoverlapping regions in the ROI (Figure 2 iv). Thus the developed confocal image suggests the localization site of the protein of interest.

Figure 2.

Represents the flow chart for generating the graphical intensity profile

(i) Region of interest (ROI) selected from the confocal image, (ii) Parameters selected in the NIS-Elements AR software image analyzer, (iii) Raw data generated in Excel format with Green, Red and Blue signal intensities and (iv) Intensity profile generated with the colored peaks.

Limitations

Although the protocol described for localization and the subcellular fraction is simple and efficient, it has some limitations. For localization studies, we found the GFP distribution in whole cell in the case of pEGFP-N1 vector (control) if allowed more than 48 h.

Troubleshooting

Problem 1

The use of coverslips with more thickness (22 mm) may hamper the visibility during imaging analysis. (Step 11 of cell counting and seeding).

Potential solution

Round coverslips with 18 mm or less than 18 mm thickness give clear visibility and hence better image acquisition.

Alternatively, Chamber slides can be used.

Problem 2

Co-transfecting two plasmids together may affect the transfection efficiency of each other’s expression (Step 2 of Co-transfection).

Potential solution

Performing transfection of each individual plasmid (as control) with the same concentration as that of co-transfected plasmids helps in the analysis of the transfection efficiency and expression of individual and co-transfected constructs.

Please see the below examples:

Combination-1: 500 ng of Plasmid X and 500 ng of Plasmid Y with 2 μL of Lipofectamine 2000 (1:2) can be co-transfected in HEK cells. The expression can be analyzed after 48 h to observe the maximum number of transfected cells.

Combination-2: 1 μg of Plasmid X and 1 μg of Plasmid Y with 4 μL of Lipofectamine 2000 (1:2) can be co-transfected in HEK cells. The expression can be analyzed after 48 h to observe the maximum number of transfected cells.

Combination-3: 1 μg of Plasmid X and 1 μg of Plasmid Y with 6 μL of Lipofectamine 2000 (1:3) can be co-transfected in HEK cells. The expression can be analyzed after 48 h to observe the maximum number of transfected cells.

The transfection of the above plasmids individually can be used as a control.

For HEK cells, the ratio 1:2 works well i.e., 1 μg to 2 μL of Lipofectamine shows good transfection efficiency i.e., up to 70%–80%.

Different cell lines have their own transfection efficiency so the plasmid concentration to Lipofectamine ratio must be standardized to obtain good transfections (see the table given below).

Also, the time of expression of each plasmid may differ, in case of HEK cells 48 h of expression give conclusive results.

Problem 3

High GFP expression may result in distribution of GFP in whole cell and might hinder the clear localization analysis (Step 5 of Co-transfection).

Potential solution

GFP fusion protein expression needs to be optimized for each cell line. Different concentrations of plasmids can be used along with Lipofectamine for optimizing the GFP distribution.

Suggested concentrations of plasmid and Lipofectamine for optimal transfections

Ratio of plasmid and Lipofectamine	Plasmid concentration (ng) and required volumes of Lipofectamine (μL)
	100 ng	500 ng	700 ng	1000 ng
1:2	0.2	0.5	1.4	2
1:3	0.3	1.5	2.1	3
1:4	0.4	2	2.8	4
1:5	0.5	2.5	3.5	5

Optimizing the transfection time according to the expression of protein of interest (12, 24 and 48 h).

Problem 4

High background in western blotting (Step 18 of western blotting).

Potential solution

Optimize the blocking buffer and incubation time. Both skimmed milk (5%–7%) or BSA (up to 5%) can be used in 1X PBST or 1X TBST.

• •Membrane can be blocked with 7% blocking buffer for 1 h 30 min at 25°C on slow rocking or with 5% blocking buffer for 2 h at 25°C or 12–14 at 4°C.

Problem 5

Appearance of nonspecific bands (Steps 19 & 22 of western blotting).

Potential solution

Optimize the primary antibody and secondary antibody dilutions and incubation times.

• •Primary antibody dilutions can be optimized by using different dilutions (1:1000, 1:3000, 1:5000) based on the expression level of protein of interest. The primary antibody can be used for 2 h at 25°C or 12–14 h at 4°C with slow rocking.
• •Secondary antibody dilutions can be used at 1:10,000 or 1:20,000 dilution at 25°C for 2 h.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Musturi Venkataramana (mvrsl@uohyd.ac.in).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate new data or code.