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Sci Rep
2017 Feb 02;7:41519. doi: 10.1038/srep41519.
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MISTIC-fusion proteins as antigens for high quality membrane protein antibodies.
Alves NS
,
Astrinidis SA
,
Eisenhardt N
,
Sieverding C
,
Redolfi J
,
Lorenz M
,
Weberruss M
,
Moreno-Andrés D
,
Antonin W
.
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Lack of high-quality antibodies against transmembrane proteins is a widely recognized hindrance in biomedical and cell biological research. Here we present a robust pipeline for the generation of polyclonal antibodies employing full-length membrane proteins as immunogens to overcome this "antibody bottleneck". We express transmembrane proteins fused to a MISTIC fragment that enhances expression of eukaryotic membrane proteins in E. coli. Purified membrane proteins are used as immunogen for rabbit injection employing standard immunizing protocols. The raised antibodies against membrane proteins of the endoplasmic reticulum and the nuclear envelope, which we use as test cases, function in a wide range of applications and are superior to ones produced against soluble domains as immunogens.
Figure 1. Antisera against full-length POM33 outperform antisera generated against a soluble fragment.(a) Schematic representation of POM33. Predicted transmembrane regions are indicated in black, the lipid bilayer in yellow. (b) 10 μg, 3 μg and 1 μg of a total membrane fraction from Xenopus egg extracts were separated on a 12% SDS-PAGE and analyzed by western blotting using antisera against full-length POM33 (antiserum A and B) in a 1:10,000 dilution and antisera against the C-terminal domain (antiserum C and D) in a 1:1,000 dilution. Molecular size markers as well as the position of POM33 are indicated. Please note that for this analysis the blot membrane was after transfer divided into four parts, which were separately incubated with the indicated antisera. For blot analysis by ECL the parts were realigned and imaged as a whole. (c) POM33 was immunprecipitated from a solubilized total membrane fraction from Xenopus egg extracts using antisera against full-length POM33 (antiserum A and B) and antisera against the C-terminal domain (antiserum C and D). Antibody bound proteins were eluated with SDS sample buffer and separated together with 10% and 30% of the corresponding starting material on a 12% SDS-PAGE. After western blotting samples were analyzed with α-POM33 antiserum B. (d) Immunofluorescence detection of POM33. Xenopus S3 cells were fixed with 2% paraformaldehyde and stained with α-POM33 antisera A and B. Samples were co-stained with the nuclear pore complex (NPC) marker mAB414 and DAPI and analyzed by confocal microscopy. Bars: 5 μm.
Figure 2. Characterization of antisera against the nuclear pore complex transmembrane protein NDC1.(a) 10 μg and 3 μg of a total membrane fraction from Xenopus egg extracts were separated on a 8% SDS-PAGE and analyzed by western blotting using antisera against full-length NDC1 (aa 1–660, antiserum A), against the N-terminal part of NDC1 which contains all six predicted transmembrane regions (aa 1–301, antiserum B and C) and against a C-terminal domain (aa 361–521, antiserum D and E, described in ref. 22). All antisera were used in a 1:1,000 dilution. Molecular size markers as well as the position of NDC1 are indicated. (b) NDC1 was immunprecipitated from a solubilized total membrane fraction from Xenopus egg extracts using antisera against full-length NDC1 (antiserum A), against the N-terminal part of NDC1 (antiserum B and C) and against a C-terminal domain (antiserum D and E). Antibody bound proteins were eluated with SDS sample buffer and separated together with 30% and 10% of the corresponding starting material on a 8% SDS-PAGE. After western blotting samples were analyzed with α-NDC1 antiserum A. Molecular size markers as well as the position of NDC1 and the detected IgG heavy chains (hc) are indicated.
Figure 3. Characterization of antisera against the type II inner nuclear membrane protein SCL1/BC08.(a) 10 μg, 3 μg and 1 μg of a total membrane fraction from Xenopus egg extracts were separated on a 15% SDS-PAGE and analyzed by western blotting using antisera against full-length SCL1/BC08 (aa 1–97, antiserum A and B) in a 1:10,000 dilution and antisera against the nucleoplasmic domain (aa 1–76, antiserum C and D) in a 1:1,000 dilution. Molecular size markers as well as the position of SCL1/BC08 are indicated. (b) SCL1/BC08 was immunprecipitated from a solubilized total membrane fraction from Xenopus egg extracts using antisera against full-length SCL1/BC08 (antiserum A and B) and antisera against the nucleoplasmic domain (antiserum C and D). Antibody bound proteins were eluated with SDS sample buffer and separated together with 30% and 100% of the corresponding starting material on a 15% SDS-PAGE. After western blotting samples were analyzed with α- SCL1/BC08 antiserum A.
Figure 4. Characterization of Calnexin antisera.(a) Schematic representation of Calnexin. The transmembrane region is indicated in black, the lipid bilayer in yellow. (b) 10 μg, 3 μg and 1 μg of a total membrane fraction from Xenopus egg extracts were separated on a 8% SDS-PAGE and analyzed by western blotting using antisera against a Calnexin fragment including its transmembrane region (aa 485–611, antiserum A and B) in a 1:10,000 dilution and antisera against the C-terminal cytoplasmic domain (aa 516–611 antiserum C and D) in a 1:1,000 dilution. Molecular size markers as well as the position of Calnexin are indicated. (c) Calnexin was immunprecipitated from a solubilized total membrane fraction from Xenopus egg extracts using antisera against a Calnexin fragment including its transmembrane region (antiserum A and B) and antisera against the C-terminal domain (antiserum C and D). Antibody bound proteins were eluated with SDS sample buffer and separated together with 10% and 30% of the corresponding starting material on a 8% SDS-PAGE. After western blotting samples were analyzed with α-Calnexin antiserum A. (d) Immunofluorescence detection of Calnexin. Xenopus S3 cells were fixed with 2% paraformaldehyde and stained with antiserum A. Samples were co-stained with the nuclear pore complex (NPC) marker mAB414 and DAPI and analyzed by confocal microscopy. Bar: 5 μm.
Figure 5. MISTIC-fusion proteins are efficient antigens for production of antibodies against membrane proteins.(a) Two antisera generated against Xenopus full-length brambleberry (BMB), two antisera against SUN1 and one antiserum against full-length Nurim were analyzed by western blotting in a 1:1000 dilution. 3 μg of a total membrane fraction from Xenopus egg extracts were separated on 12% (for BMB and Nurim) or 8% SDS-PAGEs (for SUN1). Molecular size markers as well as the position of respective proteins are indicated. Cropped images showing the whole respective lanes are shown. (b) BMB and SUN1 were immunprecipitated from a solubilized total membrane fraction from Xenopus egg extracts using antisera described in (a). Antibody bound proteins were eluated with SDS sample buffer, separated on a 12% (BMB) or 8% SDS-PAGE and analyzed with BMB serum 1 or SUN1 serum 1, respectively. Molecular size markers as well as the position of respective proteins (arrow head) and the IgG heavy chain (*) are indicated. Cropped images showing the whole respective lanes are shown. (c) Immunofluorescence detection of SUN1 and SUN2. Xenopus S3 cells were fixed with 2% PFA and stained with two antisera against SUN1 (as in a) and full-length Xenopus SUN2. Samples were co-stained with the nuclear pore complex (NPC) marker mAB414 and DAPI and analyzed by confocal microscopy. Bars: 5 μm.
Figure 6. Antisera raised against MISTIC-fusion proteins can be efficiently affinity purified.An antiserum raised against a MISTIC-fusion with the full-length transmembrane nucleoporin POM121 was affinity purified using the MISTIC-POM121 protein coupled to Affigel 10 matrix as bait. The unpurified antiserum (at a 1:1,000 dilution) as well as the affinity purified antibodies (at a 0.1 mg/ml and 0.03 mg/ml dilution) were tested by western blotting after 6% SDS-PAGE of 10 μg, 3 μg and 1 μg of a total membrane fraction from Xenopus egg extracts. Molecular size markers as well as the position of POM121 are indicated.
Figure 7. Antibodies raised against full-length POM121 and NDC1 inhibit nuclear reformation.(a) Xenopus egg extract membranes were preincubated with control or α-POM121, α-NDC1 or α-GP210 IgG, respectively, and used in nuclear assembly reactions. Membranes were stained with DiIC18 (red) and chromatin with DAPI (blue) and samples analyzed by confocal microscopy. As previously reported for antibodies against soluble domains of POM121 and NDC12230, also antibodies against full-length POM121 and NDC1 but not GP210 block formation of a closed nuclear envelope around sperm chromatin. (b) 100 randomly chosen chromatin substrates per reaction from samples from (a) were analyzed for closed nuclear envelope formation. The average of three independent experiments are shown, individual data points are indicated.
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