Preparation of antiserum and detection of proteins by Western blotting using the starlet sea anemone, Nematostella vectensis

doi:10.1038/protex.2012.057

Method Article

Preparation of antiserum and detection of proteins by Western blotting using the starlet sea anemone, Nematostella vectensis

https://doi.org/10.1038/protex.2012.057

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Among basal animals, the starlet sea anemone Nematostella vectensis (phylum Cnidaria) has emerged as a leading laboratory model organism. Herein, we describe techniques to prepare antiserum against Nematostella proteins and to detect Nematostella proteins by Western blotting of extracts from animals or tissue culture cells overexpressing Nematostella cDNAs. These protocols may also be used to complement a variety of cellular and molecular techniques being used by the increasing number of investigators who are focusing on gene regulatory systems of basal animals, such as sponges, corals, and placozoans.

Abbreviations: aa, amino acid(s); ASW, artificial sea water; BSA, bovine serum albumin; EST, expressed sequence tag; HRP, horseradish peroxidase; KLH, Keyhole Limpet hemocyanin; NGS, normal goat serum; Nv, Nematostella vectensis; PBS, phosphate-buffered saline; SDS, Sodium dodecyl sulfate; TBS, Tris-buffered saline; TBST, Tris-buffered saline-Tween-20.

Cell biology

Developmental biology

Computational biology and bioinformatics

Biological techniques

Nematostella

antiserum

Western blotting

A basic requirement for elucidating gene and protein function of any model organism is to detect specific proteins of interest. Herein, we describe protocols for detecting the expression individual proteins in extracts of the starlet sea anemone Nematostella vectensis and to verify the specificity of antiserum using transfected mammalian cells. Although these protocols were established for use with Nematostella, it is likely that they can be adapted for use with other cnidarian polyps (other anemones, corals, and hydra) or other invertebrate organisms (sponges and placozoans). Moreover, they can be used to complement a variety of other protocols that we have recently described for the investigation of gene and protein function in Nematostella^1,2.

Antisera against native Nematostella proteins, including Nv-NF-κB, Nv-IκB, and minicollagen proteins (Nv-NCol-1, Nv-NCol-3 and Nv-NCol-4), as well as a commercial antibody against 5HT serotonin receptor have been used in indirect immunofluorescent staining of anemones^3-6. Western blotting has been performed for Nv-NF-κB³, Nv-IκB⁴ and millicollagen proteins⁶, which provides an important control for specificity in immunostaining experiments in Nematostella.

The starlet sea anemone, Nematostella vectensis, is a small, geographically widespread estuarine cnidarian^7-9. Nematostella is a member of the phylum Cnidaria, which, in addition to anemones, also includes corals, jellyfish and hydra. Several genes and gene families that were previously thought to be unique to vertebrates (due to their absence from the sequenced genomes of fruit fly and soil nematodes) have been identified in Nematostella¹⁰. Such findings suggest that the genome of Nematostella has evolved in a relatively conservative fashion compared to many other invertebrates. Thus, study of gene/protein function in Nematostella is of interest from an evolutionary prospective, but is likely to also have ecological importance, given that other cnidarians (especially corals) are under threat from environmental insults.

Protein Detection

Compared to detection of RNA, the detection of proteins in or from Nematostella has been much less commonly reported^3,4,6,12. Western blotting of adult Nematostella extracts uses protocols similar to those used for detection of proteins from mammalian cell lysates^13,14. Isolation of protein extracts from Nematostella is described elsewhere¹⁵. Custom antisera developed against two Nematostella proteins, Nv-NF-κB (Fig. 1a) and Nv-IκB (Fig. 1b), were used in Western blotting to detect the relevant proteins from lysates of transfected human A293 cells and with extracts from Nematostella animals of varying stages.

• Nematostella vectensis (collection of adults and spawning techniques are described elsewhere¹⁸)

• A293 cells (ATCC, cat. no. CRL-1573)

• Bromophenol Blue (Sigma-Aldrich, cat. no. B0126)

• Bovine serum albumin (BSA; Sigma-Aldrich, cat. no. A-7888)

• ECL solution to detect HRP-conjugated antisera (SuperSignal West Dura Substrate; Pierce, cat. no. 34076)

• NaCl (4 M solution; Fisher Scientific, cat. no. BP-348-1)

• Nitrocellulose membrane (0.45 µm pore-size; Bio-Rad, cat. no. 162-0115)

• Normal goat serum (NGS; Gibco, cat. no. 16210)

• Phosphate-buffered saline (PBS)

• Powdered non-fat dry milk (LabScientific, cat. no. M0841)

• SDS ! CAUTION Causes skin, eye, and respiratory irritation.

• Tris-buffered saline (TBS)

• Tris-HCl (1 M, pH 7.4)

• Tween-20

REAGENT SET-UP

10X Phosphate-buffered Saline (PBS) Add 2.56 g NaH₂PO₄ and 11.94 g Na₂HPO₄ to 800 ml dH₂O. Add 102.2 g NaCl. Bring up to 1 liter with dH₂O. Store indefinitely at room temperature (i.e., 20-22ºC).

Tris-buffered Saline (TBS) (120 mM Tris, 150 mM NaCl)

Add 100 ml 1.2 M Tris pH 7.5 and 100 ml 1.5 M NaCl, to 800 ml dH₂O. Store indefinitely at room temperature.

TBST (120 mM Tris, 150 mM NaCl, 0.05% Tween-20)

Add 100 ml 1.2 M Tris pH 7.5, 100 ml 1.5 M NaCl, and 500 µl Tween-20 to 799.5 ml dH₂O. Store indefinitely at room temperature.

4X SDS sample buffer

Combine 20 g glycerol, 10 ml β-mercaptoethanol, 4.6 g SDS, 1.5 g Tris. Add dH₂O to _{40 ml and mix well. Adjust pH to 6.8 with 12 N HCl, and bring volume to 50 ml with dH}2_{O. Add 0.2 g (0.4% w/v) bromophenol blue dye. Mix well. Aliquot (}1 ml) and store indefinitely at 4°C.

Western Blocking Buffer (1X PBS, 5% NGS, 4% milk, 0.05% Tween-20)

Combine 40 ml dH₂O, 5 ml 10X PBS, 2.5 ml NGS, 25 μl Tween-20 and 4 g powdered milk. Mix and bring to a final volume of 50 ml with dH₂O. Make fresh for every use. CRITICAL To help reduce non-specific binding, supplement Western Blocking Buffer with 0.5 g (1%) BSA.

Table 1: Antisera used for Western blotting.

Name^a	Protocol^b	Host^c	Dilution	Source^d	Blocking Buffer^e	Duration^f
Primary Antisera
Nv-NF-κB	WB	R	1:2000	OpenBio \(custom)	TBS, 8% milk, 5% NGS, 0.05% Tw	2 h, RT
	WB	GP	1:1000	OpenBio \(custom)	TBS, 8% milk, 5% NGS, 0.05% Tw	1 h, RT
Secondary Antisera
HRP-α-R	WB	G	1:1000	CST #7074	TBS, 8% milk, 0.05% Tw	1 h, RT
HRP-α-GP	WB	G	1:20000	TF #PA1-28679	TBS, 8% milk, 5% NGS, 0.05% Tw	1 h, RT

^aAntisera conjugations: HRP, horseradish peroxidase. Secondary antisera were created against antibodies from the indicated species \(e.g., HRP-linkedanti-guinea pig is listed as HRP-α-GP).

^bDilutions and blocking buffers are specific for Immunofluorescence, IF; or Western blotting, WB.

^cAntisera host animal: G, goat; GP, guinea pig; R, rabbit.

^dSource of antisera: CST, Cell Signaling Technology; OpenBio, OpenBiosystems; TF, ThermoFisher, TF. Catalog numbers are listed below company name.

^eBlocking buffer was used to block membranes/tissue and to dilute primary antisera. Abbreviations: BSA, Bovine serum albumin; NGS, normal goat serum; TBS,Tris-buffered saline; Tx, Triton-X-100; Tw, Tween-20. All percentages are measured in \(v/v) except BSA \(w/v) and milk \(w/v).

^fBlocking was usually performed overnight at 4°C on a shaker. Duration of incubation with primary antisera: h, hours; RT, room temperature.

• Blue autoradiography film (ISC BioExpress, cat. no. F-9024-8X10)

• Dark room

• Electrophoresis power supply

• Electro-blotting apparatus

• Film developer (e.g., a Kodak RP X-OMAT)

• Heating block

• Polyacrylamide gel apparatus

• X-ray film cassette (ISC BioExpress, cat. no. SB810)

Generation of custom antiserum (~3 months)

Predict the aa sequence of your protein of interest, either from an in-house generated cDNA sequence or from the analysis of various EST databases^10,19. Note: if possible, it is good to inspect sequences from multiple cDNAs, as there is much heterogeneity in Nematostella protein coding regions, and you will want to choose a region that is highly conserved at the aa level.
Send the cDNA sequence to a commercial source for the selection of a suitable epitope. In our experience^3,4, and that of others⁶, epitopes of approximately 18 aa at the extreme C terminus generate excellent antisera.
For routine use, we have found that rabbits can be used to provide abundant, highly specific antiserum, which can be conveniently used with a number of readily available secondary antiserum. As we describe above, you are likely to want to instruct the company to use BSA as the carrier protein (because KLH generates high background with Nematostella and other marine invertebrates). Finally, you will likely need to have the antiserum purified to obtain optimal, clean Western blots.
When generated, keep antiserum at -80ºC for long-term storage. For frequent use, we generally keep small (75-100 µl) aliquots at -20ºC. These aliquots can be freeze-thawed several times without losing activity.

Preparation of A293 cell extracts expressing Nematostella protein of interest (~2 weeks)

Generate a cDNA from purified Nematostella RNA¹⁵ and subclone the cDNA into a mammalian cell overexpression vector, such as one of the pcDNA vectors (Invitrogen), using standard recombinant DNA techniques^3,4.
Transfect a 50-70% confluent 60-mm dish of A293 cells with 10 µg of the Nematostella protein expression vector as well as the empty vector as a control, according to standard techniques (e.g., see refs 3, 4).
Two days later, remove medium from cells, wash cells 2 times with PBS, and lyse the cells in 160 µl of AT buffer, as described³. Perform an assay (e.g., Bradford assay) on the clarified lysate to determine protein concentration. You are likely to have a final protein concentration of approximately 5-10 µg/µl. Store the lysate at -80ºC prior to use.

Western blotting (1 to 3 days)

Prepare Nematostella protein extract (as in refs. 3, 4).
Transfer 25 µg of Nematostella protein extract (typically ~10-20 µl) to a 1.5-ml microcentrifuge tube, and add 4X SDS sample buffer to a final concentration of 1X.
Heat sample at 95-100°C for 10 min on a heating block.
Load samples onto an SDS-polyacrylamide gel (a 10% polyacrylamide gel is suitable for routine blotting of proteins that are 40 to 100 kDa). As controls, load approximately 10-20 µg of cell lysates from both the empty vector- and pcDNA-gene vector-transfected cells.
Electrophorese samples at 80 V until the bromophenol blue passes through the stacking gel and into the resolving gel. Increase voltage to 160 V and electrophorese until the bromophenol blue reaches the bottom of the resolving gel.
Disassemble the apparatus, and discard the stacking gel.
Load the resolving gel onto the electro-blotting transfer apparatus and transfer to a nitrocellulose membrane at 100 V for 1-2 h. CRITICAL STEP High molecular weight proteins (>100 kDa) transfer better at 100 V for 4 h at 4°C. ? TROUBLESHOOTING
Disassemble the transfer apparatus and wash membrane 2 times with PBS for 1 min each.
Block membranes with 25 ml Western Blocking Buffer for at least 1 h at room temperature, although overnight at 4°C works best. CRITICAL STEP To avoid non-specific binding, 5% normal goat serum and/or 1% BSA must be added to the Western Blocking Buffer.
Probe membranes with primary antiserum diluted in Western Blocking Buffer for the appropriate length of time. The appropriate dilution of antiserum must be experimentally determined, but proteins are typically detected with dilutions that range from 1:500 to 1:10,000. A list of primary antisera that we have successfully used is presented in Table 1. ? TROUBLESHOOTING
Wash membranes 4 times with TBST for 10 min each.
Probe membranes with secondary antiserum for the appropriate length of time. A list of secondary antisera that we have successfully used is presented in Table 1.
Wash membranes 4 times with TBST for 10 min each.
Wash membrane 2 times with TBS for 5 min.
Add ECL solution and detect proteins according to manufacturer’s instructions. In a typical detection kit, both reagents (luminol and peroxide buffer) are mixed in equal volumes (0.5 ml of each) and transferred onto the nitrocellulose membrane. The ECL solution is incubated with the membrane for 5 min and then poured off.
Detect luminescence by incubating the membrane with x-ray film. This is performed in a dark room and incubation times range from 15 sec to 5 min. Once the film is exposed to the nitrocellulose membrane, it can be fixed and processed with a standard film developing machine (e.g., X-OMAT imager).
? TROUBLESHOOTING

Steps 1-4, generation of polyclonal antiserum: ~3 months

Steps 5-7, Preparation of cell extracts from animals and cells: ~ 3 days

Steps 8-23, Western blotting: The Western blotting procedure can take from 1-3 days depending on the length of incubations in Western Blocking Buffer and primary antiserum.

• Gel electrophoresis and transfer: ~ 4 h

• Blocking: 1 h to 1 day

• Incubation with primary antisera: 1 h to 1 day

• Incubation with secondary antisera: ~2 h

• Protein detection: ~1 h

Table 2: Troubleshooting

Step	Problem	Possible reason	Possible solution
23	Non-specific high molecular weight bands	Antisera cross-reacting with non-specific proteins	After electrophoresis, transfer the proteins from the gel to the membrane at 100 V for 45 min
23	Protein of interest not detected	Poor staining of primary antisera	Block membrane for 2 h at room temperature and probe with primary antiserum overnight at 4°C
23	Protein of interest detected in A293 cell extracts, but not in Nematostella extracts	Protein not extracted sufficiently from animal extracts	Boil tissue directly in SDS-sample buffer for 10 min \(i.e., do not extract protein with AT buffer)

Using this Western blotting protocol, highly specific binding can achieved to Nematostella proteins using custom antisera targeted to the C-termini of the Nematostella NF-κB and IκB proteins (Fig. 1). The protein from animal extracts should co-migrate with the protein overexpressed in A293 cells, and there should be no detected protein in the lysates from empty vector-transfected A293 cells. We optimized the protocol for different primary and secondary antisera by (1) changing the dilution of the antiserum, (2) altering the composition of the Western Blocking Buffer, and/or (3) changing the length and/or temperature of the incubation. Table 1 describes conditions that we have successfully used with two different primary antisera and two secondary antisera^3,4,12.

Other controls that might be performed are to perform a parallel Western blot using pre-immune serum for the primary antibody (no protein should be detected) or to pre-absorb antiserum with the immunogenic peptide (which should result in loss of the specific protein on Western blots). In addition, the protein may be present in soluble or insoluble extracts from animals, which will need to determined experimentally³. For example, certain cnidocyte minicollagen proteins require extensive boiling in SDS sample buffer for extraction⁶.

1 Wolenski, F.S., Layden, M.J., Martindale, M.Q., Gilmore, T.D. & Finnerty, J.R. Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis. Nat. Protoc. In press (2012).

2 Layden, M.J., Röttinger, E., Wolenski, F.S., Gilmore, T.D. & Martindale, M.Q. Microinjection techniques for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nat. Protoc. In press (2012).

3 Wolenski, F.S. et al. Characterization of the core elements of the NF-κB signaling pathway of the sea anemone Nematostella vectensis. Mol. Cell. Biol. 31, 1076-1087 (2011).

4 Wolenski, F.S., Bradham, C.A., Finnerty, J.R. & Gilmore, T.D. NF-κB is required for the development of subset of cnidocytes in the body column of the sea anemone Nematostella vectensis. Dev. Biol. 373, 205-215 (2013).

5 Marlow, H.Q., Srivastava, M., Matus, D.Q., Rokhsar, D. & Martindale, M.Q. Anatomy and development of the nervous system of Nematostella vectensis, an anthozoan cnidarian. Dev. Neurobiol. 69, 235-254 (2009).

6 Zenkert, C., Takahashi, T., Diesner, M.O. & Özbek, S. Morphological and molecular analysis of the Nematostella vectensis cnidom. PLoS ONE 6, e22725 (2011).

7 Hand, C. & Uhlinger, K. The culture, sexual and asexual reproduction, and growth of the sea anemone Nematostella vectensis. Biol. Bull. 182, 169-176 (1992).

8 Hand, C. & Uhlinger, K.R. The unique, widely distributed, estuarine sea anemone, Nematostella vectensis Stephenson: a review, new facts, and questions. Estuaries 17, 501-508 (1994).

9 Hand, C. & Uhlinger, K.R. Asexual reproduction by transverse fission and some anomalies in the sea anemone Nematostella vectensis. Invert. Biol. 114, 9-18 (1995).

10 Putnam, N. H. et al. Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317, 86-94 (2007).

11 Peterson, K.J. & Butterfield, N.J. Origin of the Eumetazoa: testing ecological predictions of molecular clocks against the Proterozoic fossil record. Proc. Natl. Acad. Sci. USA 102, 9547-9552 (2005).

12 Wolenski, F.S. et al. Two polymorphic residues account for the differences in DNA binding and transcriptional activation by NF-κB proteins encoded by naturally occurring alleles in Nematostella vectensis. J. Mol. Evol. 73, 325-336 (2011).

13 Grabski, A.C. Advances in preparation of biological extracts for protein purification. Methods Enzymol. 463, 285-303 (2009).

14 Alegria-Schaffer, A., Lodge, A. & Vattem, K. Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods Enzymol. 463, 573-599 (2009).

15 Stefanik, D.J., Wolenski, F.S., Gilmore, T.D. & Finnerty, J.R. Isolation of DNA, RNA and protein from the starlet sea anemone, Nematostella vectensis. Nat. Protoc. In press (2012).

16 Darling, J.A., Reitzel, A.M. & Finnerty, J.R. Characterization of microsatellite loci in the widely introduced estuarine anemone Nematostella vectensis. Mol. Ecol. Notes 6, 803-805 (2006).

17 Reitzel, A., Darling, J., Sullivan, J. & Finnerty, J. Global population genetic structure of the starlet anemone Nematostella vectensis: multiple introductions and implications for conservation policy. Biol. Invasions 10, 1197-1213 (2008).

18 Stefanik, D.J., Friedman, L. & Finnerty, J.R. Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis. Nat. Protoc. In Review (2012).

19 Sullivan, J.C. et al. StellaBase: the Nematostella vectensis genomics database. Nucleic Acids Res. 34, D495-499 (2006).

This research was supported by National Science Foundation grant MCB-0924749 to T.D.G and J.R.F. F.S.W. was supported by a predoctoral grant from the Superfund Basic Research Program at Boston University (5 P42 E507381) and Warren-McLeod graduate fellowships in Marine Biology.

The authors declare no competing financial interests.

supplement0.doc
Table 1 Antisera used for Western blotting.
supplement0.doc
Table 2 Troubleshooting table.

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