pTracer -EF/V5-His A, B, C - Thermo Fisher Scientific€¦ · Product description ™ pTracer -EF/V5-His is a 5.9 kb mammalian expression vector designed for visual detection of transformed

For Research Use Only. Not for use in diagnostic procedures.

pTracer™-EF/V5-His A, B, C Catalog numbers V88701, V88720

Publication Number MAN0000098

Revision A.0

Manufacturer: Life Technologies Corporation | 5791 Van Allen Way | Carlsbad, CA 92008

The information in this document is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history MAN0000098

Revision Date Description

A.0 01 August 2017 Correct the vector maps for pTracer™-EF/V5-His A, B, and C vectors, rebrand, update legal/regulatory language.

1.0 02 March 2012 Basis for this revision.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.

Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Zeocin is a trademark of CAYLA, SA.

© 2017 Thermo Fisher Scientific Inc. All rights reserved.

pTracer™-EF/V5-His A, B, C User Guide 3

Contents

Product information ........................................................................................................ 4

Product description ................................................................................................................................................ 4 Kit contents and storage ........................................................................................................................................ 4 Description of the system ...................................................................................................................................... 5 Workflow overview ............................................................................................................................................... 6

Methods .......................................................................................................................... 7

Clone into pTracer™-EF/V5-His ........................................................................................................................... 7 Transform E. coli ................................................................................................................................................... 11 Transfect mammalian cells .................................................................................................................................. 13 Detect cycle 3-GFP fluorescence ......................................................................................................................... 14 Assay for heterologous gene expression ........................................................................................................... 15 Isolate stable transfectants ................................................................................................................................... 17 Purify your recombinant protein ....................................................................................................................... 19

Appendix A: Troubleshooting ........................................................................................ 20

Appendix B: Vectors ...................................................................................................... 21

pTracer™-EF/V5-His A, B, C vectors ................................................................................................................. 21 pTracer™-EF/V5-His/lacZ control vector ......................................................................................................... 25 Human EF-1α Promoter ...................................................................................................................................... 27

Appendix C: Zeocin™ ...................................................................................................... 28

Appendix D: Ordering information ................................................................................ 29

Accessory products .............................................................................................................................................. 29

Documentation and support .......................................................................................... 31

References .............................................................................................................................................................. 32

4 pTracer™-EF/V5-His A, B, C User Guide

Product information

Product description

pTracer™-EF/V5-His is a 5.9 kb mammalian expression vector designed for visual detection of transformed E. coli cells and transfected mammalian cells. The vector yields high-level expression of the gene of interest in mammalian cells, and can be used in both transient and stable expression studies.

Kit contents and storage

Shipping and storage

Cat. No. V88701 is shipped on blue ice. Upon receipt, store both the plasmid and the Zeocin™ antibiotic at –20°C.

Cat. No. V88720 is shipped at room temperature. Upon receipt, store the plasmid at –20°C.

Kit contents This user guide is supplied with the following products.

Cat. No. Reagent Composition Amount

V88701 pTracer™-EF/V5-His A, B & C 0.5 μg/μL in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 in a total volume of 40 μL

20 μg

pTracer™-EF/V5-His/lacZ control plasmid

0.5 μg/μL in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 in a total volume of 40 μL

20 μg

Zeocin™ antibiotic 100 mg/mL, 8 × 1.25 mL 1 g

V88720 pTracer™-EF/V5-His A, B & C 0.5 μg/μL in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 in a total volume of 40 μL

20 μg

pTracer™-EF/V5-His/lacZ control plasmid

0.5 μg/μL in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 in a total volume of 40 μL

20 μg


Description of the system

pTracer™-EF/V5-His vector

pTracer™-EF/V5-His vector is supplied in three reading frames (A, B and C) to facilitate in-frame cloning with a C-terminal detection and purification tag. The features of the pTracer™-EF/V5-His vector are summarized below. For more information, see pages 21–24.

• Cycle 3-GFP, an improved GFP (Green Fluorescent Protein) gene (Crameri et al., 1996) for noninvasive in vivo detection, fused to the Zeocin™ resistance gene.

• Zeocin™ resistance gene for selection in both E. coli and mammalian cells (see page 28).

• Human cytomegalovirus immediate-early (CMV) promoter drives the expression of the cycle 3-GFP-Zeocin™ resistance gene fusion in mammalian cells.

• Human elongation factor 1α (hEF-1α) promoter permits overexpression of your recombinant protein in a broad range of mammalian cell types (Goldman et al., 1996; Mizushima and Nagata, 1990) (see page 27).

• C-terminal peptide containing the V5 epitope for detection of your fusion protein and a polyhistidine tag (6xHis) for purification on metal-chelating resin, such as ProBond™.

pTracer™-EF/V5-His vector

pTracer™-EF/V5-His vector kits include a positive control vector, pTracer™-EF/V5-His/lacZ, to test for proper expression and detection. See page 25 for more information.

Description of cycle 3-GFP

The cycle 3-GFP gene used in this vector is described by Crameri et al., 1996. In this paper, the codon usage of GFP was optimized for expression in E. coli, followed by three cycles of DNA shuffling. A mutant form of GFP was selected that gave the greatest fluorescence signal in mammalian cells. Cycle 3-GFP has the following characteristics:

• Excitation and emission maxima that are the same as wild-type GFP (395 nm and 478 nm for primary and secondary excitation, respectively, and 507 nm for emission)

• High solubility in E. coli for visual detection of transformed cells

• >40-fold increase in fluorescent yield over wild-type GFP

• The cycle 3-GFP gene is fused to the Zeocin™ resistance marker to correlate GFP fluorescence with resistance to Zeocin™.


Workflow overview

The table below outlines the basic steps needed to clone and express your gene of interest in pTracer™-EF/V5-His and to visually detect transformed or transfected cells.

Step Action Page

1 Develop a cloning strategy to ligate your gene of interest into one of the pTracer™-EF/V5-His vectors. Use the diagrams of the multiple cloning sites on pages 8–10 to assist you.

7

2 Transform E. coli and select transformants. 11

3 Optional: Visually detect transformed cells using a transilluminator or hand-held UV lamp.

12

4 Analyze transformants for the presence and orientation of the insert.

12

5 Isolate pure plasmid DNA for mammalian transfection. 13

6 Transfect your mammalian cell line. 13

7 Assay for fluorescence. 14

8 Assay for optimal expression of your gene. 15

9 Optional: Select for stable cell lines using Zeocin™. 17

10 Purify your recombinant protein 19


Methods

Clone into pTracer™-EF/V5-His

Introduction Diagrams are provided on the following pages to help you ligate your gene of interest into pTracer™-EF/V5-His. General considerations are listed below for additional information. If you wish to use Zeocin™ for selection in E. coli, see page 11.

General molecular biology techniques

For help with DNA ligations, E. coli transformations, restriction enzyme analysis, purification of single-stranded DNA, DNA sequencing, and DNA biochemistry, see Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).

Maintenance of pTracer™-EF/V5-His

To propagate and maintain pTracer™-EF/V5-His, use the supplied 0.5 μg/μL stock solution in TE buffer, pH 8.0 to transform a recA, endA E. coli strain such as TOP10, TOP10F´, DH5α™, JM109, or equivalent.

Do not use E. coli strains containing the complete Tn5 transposon (page 11).

Select transformants on 50-100 µg/mL ampicillin or 25–50 µg/mL Zeocin™ (be sure to use Low Salt LB medium when selecting on Zeocin™; see page 11).

Prepare a glycerol stock for long-term storage (page 12). Cloning considerations

Your insert should contain a Kozak translation initiation sequence for proper initiation of translation (Kozak, 1987; Kozak, 1990; Kozak, 1991). An example of a Kozak consensus sequence is provided below. Note that other sequences are possible (see references above), but the G or A at position –3 and the G at position +4 are the most critical (shown in bold). The ATG initiation codon is shown underlined.

(G/A)NNATGG

pTracer™-EF/V5-His A, B and C are C-terminal fusion vectors. To express your gene as a recombinant fusion protein, you must clone your gene in frame with the C-terminal V5-His peptide tag. The vector is supplied in three reading frames to facilitate cloning. See pages 8–10 to develop a cloning strategy.

If you wish to express your protein without the C-terminal peptide, be sure to include a stop codon.


Clone into pTracer™-EF/V5-His A

The graphic below shows the multiple cloning site of pTracer™-EF/V5-His A. Restriction sites are labeled to indicate the cleavage site. Note that there is a potential stop codon between the SpeI and the first BstXI site. The boxed nucleotides indicate the variable region. The multiple cloning site has been confirmed by sequencing and functional testing. The vector sequence is available for download at thermofisher.com or from Technical Support (page 31).

*Note: There are two Bst XI sites.

http://www.thermofisher.com/


Clone into pTracer™-EF/V5-His B

The graphic below shows the multiple cloning site of pTracer™-EF/V5-His B. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region. The multiple cloning site has been confirmed by sequencing and functional testing. The vector sequence is available for download at thermofisher.com or from Technical Support (page 31).

*Note: There are two Bst XI sites



Clone into pTracer™-EF/V5-His C

The graphic below shows the multiple cloning site of pTracer™-EF/V5-His C. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region. The multiple cloning site has been confirmed by sequencing and functional testing. The vector sequence is available for download at thermofisher.com or from Technical Support (page 31).

*Note: There are two Bst XI sites



Transform E. coli

Transformation method

You can use any method to transform your ligation mixture into a competent recA, endA E. coli strain (TOP10, TOP10F´ or similar). You can select transformants on ampicillin (50–100 µg/mL) or Zeocin™ (25–50 µg/mL). If you elect to use Zeocin™, read the information in this section before proceeding.

E. coli strain Many E. coli strains are suitable for the growth of this vector including TOP10F´,

DH5αF´, and JM109. We recommend that you propagate vectors containing inserts in E. coli strains that are recombination deficient (recA) and endonuclease A deficient (endA).

For your convenience, TOP10F´ is available as chemically competent or electrocompetent cells for purchase (page 29).

IMPORTANT! If you plan to use Zeocin™ to select transformants, do not use any E. coli strain that contains the complete Tn5 transposable element (i.e., DH5F´IQ™, SURE, SURE2). This transposon encodes a ble (bleomycin) resistance gene which will confer resistance to Zeocin™, preventing selection of colonies containing the pTracer™-EF/V5-His vector.

Prepare Low Salt LB medium with Zeocin™

If you wish to select bacterial transformants using Zeocin™, use Low Salt LB medium. For maximal activity of Zeocin™, the salt concentration of LB medium must remain low (<90 mM) and the pH must be 7.5. Prepare LB broth and plates using the following recipe.

Failure to lower the salt content of your LB medium will result in non-selection due to inactivation of the drug.

Low Salt LB Medium:

10 g Tryptone 5 g NaCl 5 g Yeast Extract

1. Combine the dry reagents above and add deionized, distilled water to 950 mL. Adjust pH to 7.5 with 1 N NaOH. Bring the volume up to liter. For plates, add 15 g/L agar before autoclaving.

2. Autoclave on liquid cycle at 15 psi and 121°C for 20 minutes.

3. Allow the medium to cool to at least 55°C before adding the Zeocin™ to 25 µg/mL final concentration.

4. Store plates at 4°C in the dark. Plates containing Zeocin™ are stable for 1–2 weeks.


Detect E. coli transformants

After overnight incubation, you can detect transformed E. coli by placing the plates on a transilluminator or observing them with a hand-held UV lamp set on the long UV wavelength setting. Transformed colonies can be easily detected by a bright green fluorescence. Select 10–20 clones and analyze for the presence and orientation of your insert.

Note: Fluorophore formation in E. coli grown at 37°C under aerobic conditions has a T1/2 of about 95 minutes. It has been reported that E. coli containing cycle 3-GFP grow 2- to 3-fold faster than E. coli containing wild-type GFP. This is presumably because of the reduced toxicity of the soluble cycle 3-GFP (Crameri et al., 1996).

Sequence your cloned construct

We recommend that you sequence your construct to confirm the presence of a Kozak sequence and in-frame cloning of your gene with the C-terminal tag. The T7 Promoter Primer and BGH reverse primer sequences can be used to sequence across the multiple cloning site. Refer to the diagram on pages 8–10 for the sequence and location of the primer binding sites.

For your convenience, we offer the T7 Promoter Primer (see page 29) as well as a custom primer synthesis service. For more information, go to thermofisher.com or contact Technical Support (page 31).

Prepare a glycerol stock

Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage at –80°C. It is also a good idea to keep a DNA stock of your plasmid at –20°C.

1. Streak the original colony out on an LB plate containing 50 μg/mL ampicillin or a low salt LB plate containing 25 μg/mL Zeocin™. Incubate the plate at 37°C overnight.

2. Isolate a single colony and inoculate 1–2 mL of LB containing 50 μg/mL ampicillin or 1–2 mL Low Salt LB containing 25 μg/mL Zeocin™.

3. Grow the culture to mid-log phase (OD600 = 0.5–0.7).

4. Mix 0.85 mL of culture with 0.15 mL of sterile glycerol and transfer to a cryovial.

5. Store at –80°C.



Transfect mammalian cells

Overview At this point, you should have a positive clone with your gene of interest cloned in frame with the C-terminal V5-His tag (if desired). The next step is to isolate clean DNA and transfect your cell line. Before transfecting your cells, read “Detect cycle3-GFP fluorescence” (page 14) for information about using fluorescence to detect the transfected cells.

After mammalian transfection, you can harvest the cells and assay for transient expression (page 15), or split the cells and select for stable transformants using the appropriate concentration of Zeocin™ (page 17).

Plasmid preparation for transfection

Plasmid DNA must be of high quality and free of contaminants such as phenol and sodium chloride. Contaminated DNA can be toxic to many cell lines and salt will interfere with lipids, decreasing the transfection efficiency. We recommend isolating DNA using the PureLink™ HiPure Miniprep Kit or the PureLink™ HiPure Midiprep Kit (page 29), or CsCl gradient centrifugation.

Transfection methods

For established cell lines (e.g. HeLa), consult original references or the supplier of your cell line for the optimal method of transfection. It is recommended that you follow exactly the protocol for your cell line. Pay particular attention to medium requirements, when to pass the cells, and at what dilution to split the cells. Further information is provided in Current Protocols in Molecular Biology (Ausubel et al., 1994).

There are a variety of methods available for mammalian cell transfection. Methods for transfection include calcium phosphate (Chen and Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989), and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). We offer the Calcium Phosphate Transfection Kit (page 27) and a large variety of reagents for mammalian transfection. For more information on the available reagents, go to thermofisher.com or contact Technical Support (page 31).



Detect cycle 3-GFP fluorescence

Overview After transfecting your cells, you can monitor for fluorescence of cycle 3-GFP using fluorescence microscopy. Only transfected cells will emit a green fluorescent signal upon illumination, and the fluorescence can be used to estimate the transfection efficiency.

Detect fluorescence To detect fluorescent cells, it is important to pick the best filter set to optimize

detection. The primary excitation peak of cycle 3-GFP is at 395 nm. There is a secondary excitation peak at 478 nm. Excitation at these wavelengths yields a fluorescent emission peak with a maximum at 507 nm (see figure).

Use of the appropriate filter set ensures that the optimal regions of the cycle 3-GFP spectra are excited and passed (emitted). For example, the FITC filter set that we use excites cycle 3-GFP with light from 460 to 490 nm, which covers the secondary excitation peak. The filter set passes light from 515 to 550 nm, allowing the detection of most of the GFP fluorescence. Standard FITC filters easily suit most purposes; however, it is important to keep in mind that the fluorescence is affected by the sample assayed and the filter you choose.

For general information about GFP fluorescence and detection, refer to Current Protocols in Molecular Biology, pages 9.7.22 to 9.7.28 (Ausubel et al., 1994).

Detect transfected cells

After transfection, allow the cells to recover for 24–48 hours before assaying for fluorescence.

Note: Most media fluoresce because of the presence of riboflavin (Zylka and Schnapp, 1996) and may interfere with detection of cycle 3-GFP fluorescence. Medium can be removed and replaced with PBS to alleviate this problem. Be sure to replace PBS with fresh medium if you wish to continue growing the cells.

You can use fluorescence to estimate transfection efficiency and normalize any subsequent assay for your gene of interest. Estimate the total number of cells before assaying for fluorescence. Then check your plate for fluorescent cells.

You can incubate your cells further to optimize expression of your gene of interest.


Assay for heterologous gene expression

Overview The expression of cycle 3-GFP does not necessarily correlate with expression of your gene. We recommend that you perform a time course to determine the optimal time to assay for transient expression of the gene of interest (e.g. 24, 48, 72 hours etc. after transfection). Optimal times can vary from 24 to 96 hours from the time of transfection depending on cell line. We recommend that you also do a transfection with pTracer™-EF/V5-His/lacZ as a positive control for transfection and expression (page 16).

Detect recombinant fusion proteins

To detect expression of your fusion protein from pTracer™-EF/V5-His, you can use the Anti-V5 antibodies or the Anti-His(C-term) antibodies (available separately; see page 29) or an antibody to your protein of interest. In addition, the Positope™ Control Protein is available for use as a positive control for detection of fusion proteins containing a V5 epitope and a polyhistidine (6xHis) tag (page 29). WesternBreeze™ Chromogenic Kits and WesternBreeze™ Chemiluminescent Kits are available for purchase to facilitate detection of antibodies by colorimetric or chemiluminescent methods. For more information, go to thermofisher.com or contact Technical Support (page 31).

Prepare cell lysates To detect the fusion protein by Western blot, you will need to prepare a cell lysate

from transfected cells. To lyse cells:

1. Wash cell monolayers (~106 cells) twice with phosphate-buffered saline (PBS).

2. Scrape cells into 1 mL PBS and pellet the cells at 1500 × g for 5 minutes.

3. Resuspend in 50 µL Cell Lysis Buffer (see recipe on next page). Other lysis buffers may also be suitable. Vortex.

4. Incubate cell suspension at 37°C for 10 minutes to lyse the cells.

Note: You may prefer to lyse the cells at room temperature or on ice if degradation of your protein is a potential problem.

5. Vortex the cell lysate, place on ice for 2 minutes and centrifuge at 10,000 × g for 10 minutes to pellet nuclei. Transfer the supernatant to a fresh tube.

6. Assay the supernatant for protein concentration.

Note: Do not use protein assays utilizing Coomassie Blue or other dyes. NP-40 interferes with the binding of the dye with the protein.

7. Add SDS-PAGE sample buffer to a final concentration of 1X and boil the sample for 5 minutes.

8. Load 20 µg of lysate onto an SDS-PAGE gel and electrophorese. Use the appropriate percentage of acrylamide to resolve your fusion protein.

9. After electrophoresis, perform a western blot using your preferred method.

Note: The C-terminal tag adds about 5 kDa to the size of your protein. Additional amino acids may be added to your protein depending on the sites used to clone the gene of interest.



Prepare cell lysis buffer

50 mM Tris, pH 8.0

150 mM NaCl

1% Nonidet P-40

1. This solution can be prepared from the following common stock solutions. For 100 mL, combine:

1 M Tris base 5 mL 5 M NaCl 3 mL Nonidet P-40 1 mL

2. Bring the volume up to 90 mL with deionized water and adjust the pH to 8.0 with HCl.

3. Bring the volume up to 100 mL. Store at room temperature.

Note: You can add protease inhibitors at the following concentrations:

1 mM PMSF 1 µg/mL Pepstatin

1 µg/mL Leupeptin Positive control pTracer™-EF/V5-His/lacZ is provided as a positive control vector for mammalian

transfection and expression (page 25). It may be used to optimize transfection conditions for your cell line and test for expression. The gene encoding β-galactosidase is expressed in mammalian cells with a C-terminal V5-His tag (MW = 121 kDa). A successful transfection will result in β-galactosidase expression that can be easily assayed.

Assay for ϐ-galactosidase activity

A successful transfection of the control vector pTracer™-EF/V5-His/lacZ results in expression of β-galactosidase. You may assay for β-galactosidase expression by activity assay using cell-free lysates (Miller, 1972) or by staining the cells for activity. We offer the β-Gal Assay Kit and the β-Gal Staining Kit for fast and easy detection of β-galactosidase expression (page 29).

Protein purification You will need 5 × 106 to 1 × 107 transfected cells for purification of your protein

on a 2 mL ProBond™ column (or other metal-chelating column). Refer to the manufacturer's instructions before attempting to purify your fusion protein. To prepare cells for lysis, see page 19.


Isolate stable transfectants

Overview To generate a stable cell line expressing your protein, you need to determine the minimum concentration of Zeocin™ needed to prevent growth of untransfected cells. This concentration of drug will be used to select for stable transfectants. Typically, concentrations between 50 and 1000 µg/mL Zeocin™ are sufficient to kill the untransfected host cell line, with the average being 250–400 µg/mL. In general, it takes 2–6 weeks to select foci with Zeocin™ depending on the cell line. You want to be able to isolate several foci to expand into stable cell lines. Be sure to use buffered medium as Zeocin™ is sensitive to changes in pH.

Effect of Zeocin™ on sensitive and resistant cells

The Zeocin™ method of killing is quite different from neomycin and hygromycin. Cells may not round up and detach from the plate. Sensitive cells may exhibit the following morphological changes upon exposure to Zeocin™:

• Vast increase in size (similar to the effects of cytomegalovirus infecting permissive cells)

• Abnormal cell shape

• Presence of large empty vesicles in the cytoplasm (breakdown of the endoplasmic reticulum and Golgi apparatus, or other scaffolding proteins)

• Breakdown of plasma and nuclear membrane (appearance of many holes in these membranes)

Eventually, cells sensitive to Zeocin™ completely break down and only cellular debris remain.

Zeocin™-resistant cells should continue to divide at regular intervals to form distinct colonies. There should not be any distinct morphological changes in Zeocin™-resistant cells when compared to cells not under selection with Zeocin™.

Determine Zeocin™ sensitivity

To determine the minimal concentration of Zeocin™ required to prevent growth of the parental cell line, use the protocol below:

1. Seed cells (2 × 105 cells per 60 mM plate) for each concentration to be tested and allow cells to adhere. Grow for 24 hours.

2. Remove the medium and add medium containing varying concentrations of Zeocin™ (e.g. 0, 50, 100, 200, 400, 600, 800 and 1000 µg/mL) to each plate.

3. Replenish the selective media every 3–4 days, and observe the percentage of surviving cells.

4. Count the number of viable cells at regular intervals to determine the appropriate concentration of Zeocin™ that prevents growth. Select the appropriate Zeocin™ concentration that prevents growth of cells in 10–14 days.

Note: For more information on Zeocin™, see page 28.


Linearize vector for stable integration

To obtain stable transfectants, you may choose to linearize your vector before transfection. While linearizing your vector may not improve your chances of obtaining stable transfectants, it ensures that the vector does not integrate in a way that disrupts the gene of interest. The following table lists unique sites that may be used to linearize your construct prior to transformation. Other restriction sites are possible. Note that for the enzymes listed, the cleavage site is indicated for versions A, B, and C of pTracer™-EF/V5-His. Be sure that your insert does not contain the restriction enzyme site you wish to use to linearize your vector.

Enzyme Restriction site (bp) (A, B, C) Location Supplier

Ahd I 5148 (A) 5152 (B) 5143 (C) Ampicillin gene New England Biolabs

Fsp I 5370 (A) 5374 (B) 5365 (C) Ampicillin gene Amersham, New England Biolabs

Nru I 331 (A, B, C) Upstream of hEF1α promoter Thermo Fisher Scientific

Pvu I 5518 (A) 5522 (B) 5513 (C) Ampicillin gene Thermo Fisher Scientific

Sap I 4151 (A) 4155 (B) 4147 (C) Between SV40 poly A and pMB1 New England Biolabs

Sca I 5628 (A) 5632 (B) 5623 (C) Ampicillin gene Thermo Fisher Scientific

Ssp I 4 (A, B, C) Upstream of hEF1α promoter Thermo Fisher Scientific

Select stable integrants

After you have determined the appropriate Zeocin™ concentration to use (page 17), you can generate a stable cell line with your construct. You can use fluorescence to monitor the development of foci and ensure a homogeneous population of cells (page 14).

1. Transfect 106 cells in a 100 mM plate with 20 µg of vector using the desired protocol and plate onto 100 mM culture plates. Remember to include a plate of untransfected cells as a negative control.

2. 24 hours after transfection, wash the cells one time with 1X PBS and add fresh medium to the cells.

3. 48 hours after transfection, split the cells into four to eight 100 mM plates such that the cells are no more than 25% confluent. By using different dilutions, you will have a better chance at identifying and selecting foci. Incubate the cells overnight.

4. Add fresh medium containing Zeocin™ at the pre-determined concentration required for your cell line.

5. Feed the cells with selective medium every 3–4 days until foci can be identified. Use fluorescence to monitor developing foci for expression.

6. Pick the colonies using cloning rings (if the colonies are isolated and large enough) or a pipette tip (if the colonies are small) and transfer to 96- or 48-well plates. Grow the cells to near confluency before expanding.

7. Test clones for expression of your protein. You can further expand the positive clones and re-test them to confirm expression.


Purify your recombinant protein

Prepare cells for lysis

Use the following procedure to prepare cells for lysis prior to purification of your protein on ProBond™ resin. You will need 5 × 106 to 107 cells for purification of your protein on a 2 mL ProBond™ column. For detailed instructions, refer to the ProBond™ Purification user guide, available for download at thermofisher.com.

1. Seed cells (from a stable cell line) in either five T-75 flasks or two to three T-175 flasks.

2. Grow the cells in selective medium until they are 80–90% confluent.

3. Harvest the cells by treating with trypsin-EDTA for 2–5 minutes or by scraping the cells in PBS.

4. Inactivate the trypsin by diluting with fresh medium (if necessary) and transfer the cells to a sterile microcentrifuge tube.

5. Centrifuge the cells at 1500 × g for 5 minutes. Resuspend the cell pellet in PBS.

6. Centrifuge the cells at 1500 × g for 5 minutes. You can lyse the cells immediately or freeze in liquid nitrogen and store at –70°C until needed.

Lyse cells If you are using ProBond™ resin, refer to the ProBond™ Purification manual for

details about sample preparation for chromatography. Ordering information for ProBond™ is provided on page 30.

If you are using other metal-chelating resin, refer to the manufacturer's instruction for recommendations on sample preparation.



Appendix A: Troubleshooting

The pTracer™-EF/V5-His vector was tested in COS cells. Although expression of your gene may vary from cell line to cell line, we provide below some basic guidelines to troubleshoot any unexpected result. For additional assistance, contact Technical Support (page 31).

Low or no fluorescence

1. Check your original E. coli transformant by growing a 2–5 mL culture to saturation and assay for fluorescence by using a hand-held UV lamp or holding it over the transilluminator. If you detect fluorescence, your construct is fine, and you need to consider the other possibilities below.

2. High background fluorescence due to riboflavin in the culture medium. Replace medium with 1X PBS to eliminate background fluorescence.

3. A filter set was used that did not allow excitation at the optimal wavelength or permit detection of the emitted fluorescence. Check the filter set you are using.

4. Transfection efficiency is too low to allow detection of transfected cells. Optimize your transfection conditions or try another method.

5. Expression of cycle 3-GFP may be low depending on the cell line used. In COS cells, full fluorescence was observed 48 hours post-transfection.

No transient expression

Make sure there is an initiation codon in a proper Kozak consensus sequence (page 7) for eukaryotic expression.

No stable expression

1. Confirm integration of your construct by either isolating genomic DNA and performing a Southern blot or by PCR analysis to see if your gene is present.

2. Confirm transcription by isolating mRNA and performing a Northern or RT-PCR to test for expression of your gene.

Note: Be sure that the plasmid is not being maintained episomally.

3. Be sure and isolate at least 50 independent foci as the location of integration may affect expression of both promoters.


Appendix B: Vectors

pTracer™-EF/V5-His A, B, C vectors

Map of pTracer™-EF/V5-His A

The following figure summarizes the features of the pTracer™-EF/V5-His A vector (5948 bp). The vector sequence of pTracer™-EF/V5-His A is available for download at thermofisher.com or from Technical Support (page 31).

http://www.lifetechnologies.com/


Map of pTracer™-EF/V5-His B

The following figure summarizes the features of the pTracer™-EF/V5-His B vector (5952 bp). The vector sequence of pTracer™-EF/V5-His B is available for download at thermofisher.com or from Technical Support (page 31).



Map of pTracer™-EF/V5-His C

The following figure summarizes the features of the pTracer™-EF/V5-His C vector (5943 bp). The vector sequence of pTracer™-EF/V5-His C is available for download at thermofisher.com or from Technical Support (page 31).



Features of pTracer™-EF/V5-His A, B, C vectors

Features of pTracer™-EF/V5-His A (5948 bp), pTracer™-EF/V5-His B (5952 bp) and pTracer™-EF/V5-His C (5943 bp) are described in the following table. All features have been functionally tested. The multiple cloning site has been tested by restriction analysis.

Features Function

Human elongation factor 1 (hEF-1α) promoter

Provides high-level expression of the gene of interest in a broad range of mammalian cell lines (Goldman et al., 1996; Mizushima and Nagata, 1990).

T7 promoter priming site Allows sequencing of insert.

Multiple cloning site (MCS) in three reading frames

Permits insertion of gene of interest for expression and facilitates cloning in frame with the C-terminal V5 epitope and polyhistidine tag.

V5 epitope (Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr)

Allows detection of your recombinant protein with the Anti-V5 and Anti-V5-HRP antibodies (Southern et al., 1991) (see page 29 for ordering).

C-terminal polyhistidine tag Permits purification of your recombinant protein on metal-chelating resin such as ProBond™. In addition, the C-terminal polyhistidine tag is the epitope for the Anti-His(C-term) and the Anti-His(C-term)-HRP antibodies (see page 29 for ordering).

Bovine growth hormone (BGH) polyadenylation signal

Efficient transcription termination and poly-adenylation of mRNA (Goodwin and Rottman, 1992).

Human cytomegalovirus (CMV) immediate-early promoter/enhancer

Permits efficient, high-level expression of the cycle 3-GFP-Zeocin™ resistance gene fusion (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987).

EM7 promoter Permits efficient expression of the cycle 3-GFP-Zeocin™ resistance gene fusion in E. coli.

Cycle 3-GFP-Zeocin™ fusion Visual detection of transformed or transfected cells using fluorescent microscopy. Selection of transformants in E. coli and stable mammalian cell lines.

SV40 polyadenylation signal mRNA stability and transcription termination.

pUC origin Replication, maintenance, and high copy number in E. coli.

Ampicillin resistance gene (β-lactamase)

Selection of vector in E. coli.


pTracer™-EF/V5-His/lacZ control vector

Description pTracer™-EF/V5-His/lacZ is a 8980 bp control vector containing the gene for β-galactosidase. The vector was constructed by ligating a Kpn I/Pme I fragment containing the lacZ gene fused to the V5 epitope and polyhistidine tag into the pTracer™-EF/V5-His A vector.


Map of pTracer™-EF/V5-His/lacZ

The figure below summarizes the features of the pTracer™-EF/V5-His/lacZ control vector. The vector sequence for pTracer™-EF/V5-His/lacZ is available for download at thermofisher.com or by contacting Technical Support (page 31).



Human EF-1α Promoter

Description The diagram below shows the features of the hEF-1α promoter used in the pTracer™-EF/V5-His vectors (Mizushima and Nagata, 1990). Features are marked as per Uetsuki et al., 1989.


Appendix C: Zeocin™

Overview Zeocin™ is a member of the bleomycin/phleomycin family of antibiotics isolated from Streptomyces (Berdy, 1980). Zeocin™ and the resistance gene (Sh ble) can be used for selection in mammalian cells (Mulsant et al., 1989); plants (Perez et al., 1989); yeast (Baron et al., 1992); and prokaryotes (Drocourt et al., 1990). It is particularly well-suited for selection of mammalian stable cell lines.

Chemical properties of Zeocin™

Zeocin™ is a formulation of phleomycin D1, a basic, water-soluble, copper-chelated glycopeptide isolated from Streptomyces verticillus. The presence of copper gives the solution its blue color. This copper-chelated form is inactive. When the antibiotic enters the cell, the copper cation is reduced from Cu2+ to Cu1+ and removed by sulfhydryl compounds in the cell. Upon removal of the copper, Zeocin™ is activated and will bind DNA and cleave it, causing cell death.

Molecular weight, formula, and structure

The formula for Zeocin™ is C55H85 O21N20S2Cu-HCl and the molecular weight is 1,525. The structure of Zeocin™ is shown below (Berdy, 1980).

How to handle Zeocin™

• High salt and acidity or basicity inactivates Zeocin™. Therefore, we recommend that you reduce the salt in bacterial medium and adjust the pH to 7.5 to keep the drug active (see page 8).

• Store Zeocin™ at –20°C and thaw on ice before use.

• Zeocin™ is light sensitive. Store drug, plates and medium containing drug in the dark.

• Wear gloves, a laboratory coat, and safety glasses or goggles when handling solutions containing Zeocin™.

• Zeocin™ is toxic. Do not ingest or inhale solutions containing the drug.


Appendix D: Ordering information

Accessory products

Additional products The products listed in this section are intended for use with pTracer™-EF/V5-His and are available separately for purchase.

Product Quantity Cat. No.

Electrocomp™ TOP10F´ 5 × 80 µL C66555

Ultracomp™ TOP10F´ (chemically competent E. coli) 5 × 300 µL C66503

One Shot™ TOP10F´ (chemically competent E. coli) 21 × 50 µL C303003

Calcium Phosphate Transfection Kit 75 reactions K278001

Zeocin™ 1 g R25001

5 g R25005

T7 Promoter Primer 2 μg N56002

β–Gal Assay Kit 1 kit K145501

β–Gal Staining Kit 1 kit K146501 Detection of fusion protein

A number of antibodies are available for purchase to detect expression of your fusion protein from pTracer™-EF/V5-His. Horseradish peroxidase (HRP)-conjugated antibodies allow one-step detection in western blots using colorimetric or chemiluminescent detection methods. The amount of antibody supplied is sufficient for 25 westerns.

Antibody Epitope Cat. No.

Anti-V5 Detects 14 amino acid epitope derived from the P and V proteins of the paramyxovirus, SV5 (Southern et al., 1991): GKPIPNPLLGLDST

R96025

Anti-V5-HRP Same as Anti-V5 antibody R96125

Anti-His(C-term) Detects the C-terminal polyhistidine (6xHis) tag (requires the free carboxyl group for detection) (Lindner et al., 1997): HHHHHH-COOH

R93025

Anti-His(C-term)-HRP Same as Anti-His(C-term) antibody R93125

Positope™ Control Protein (5 μg)

— R90050


Purification of fusion protein

The polyhistidine (6xHis) tag allows purification of the recombinant fusion protein using metal-chelating resins such as ProBond™.

Product Quantity Cat. No.

ProBond™ Purification System

12 mL precharged ProBond™ resin, 6 polypropylene columns, and buffers for native and denaturing purification

K85001

ProBond™ Purification System with Anti-V5-HRP Antibody

1 Kit (same as ProBond™ Purification System plus 50 μL of antibody) The amount of antibody supplied is sufficient for 25 westerns.

K85401

ProBond™ Nickle-Chelating Resin

50 mL R80101

150 mL R80115

Purification Columns 50 polypropylene columns R64050


Documentation and support

Obtaining support

Technical Support For the latest services and support information for all locations, go to www.thermofisher.com.

At the website, you can:

• Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support (thermofisher.com/support)

• Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents

• Obtain information about customer training

• Download software updates and patches

Safety Data Sheets (SDS)

Safety Data Sheets (SDSs) are available at www.thermofisher.com/sds.

IMPORTANT! For the SDSs of chemicals not distributed by Thermo Fisher Scientific, contact the chemical manufacturer.

Limited Product Warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale found on Life Technologies’ website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support.


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Berdy, J. (1980) Bleomycin-Type Antibiotics. In Amino Acid and Peptide Antibiotics, J. Berdy, ed. (Boca Raton, FL: CRC Press), pp. 459-497.

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Drocourt, D., Calmels, T. P. G., Reynes, J. P., Baron, M., and Tiraby, G. (1990). Cassettes of the Streptoalloteichus hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance. Nucleic Acids Res. 18, 4009.

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Goodwin, E. C., and Rottman, F. M. (1992). The 3´-Flanking Sequence of the Bovine Growth Hormone Gene Contains Novel Elements Required for Efficient and Accurate Polyadenylation. J. Biol. Chem. 267, 16330-16334.

Goldman, L.A., Cutrone, E.C., Kotenko, S.V., Krause, C.D., and Langer, J.A. (1996). Modifications of Vectors pEF-BOS, pcDNA1, and pcDNA3 Resut in Improved Convenience and Expression. BioTechniques 21, 1013-1015.

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Mulsant, P., Tiraby, G., Kallerhoff, J., and Perret, J. (1989). Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells. Somat. Cell Mol. Genet. 14, 243-252.

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pTracer -EF/V5-His A, B, C - Thermo Fisher Scientific€¦ · Product description ™ pTracer -EF/V5-His is a 5.9 kb mammalian expression vector designed for visual detection of transformed