Anti-SNRP70/U1-70K antibody

• WB

Unknown

Western blot - Anti-SNRP70/U1-70K antibody (AB83306)

The 63 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to Human U1 small nuclear ribonucleoprotein 70 kDa.

All lanes:

Western blot - Anti-SNRP70/U1-70K antibody (ab83306) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 3:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 4:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Lane 5:

Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 6:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 7:

SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 52 kDa

Observed band size: 63 kDa

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Exposure time: 2min

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SNRP70/U1-70K antibody (AB83306)

IHC image of SNRP70/U1-70K antibody staining in a section of formalin-fixed paraffin-embedded normal human colon* performed on a Leica BOND^TM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab83306, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SNRP70/U1-70K antibody (AB83306)

ab83306 staining SNRP70/U1-70K in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab83306 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Unknown

Western blot - Anti-SNRP70/U1-70K antibody (AB83306)

All lanes:

Western blot - Anti-SNRP70/U1-70K antibody (ab83306) at 1 µg/mL

Lane 1:

Testis (Rat) Tissue Lysate at 10 µg

Lane 2:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Lane 3:

Testis (Mouse) Tissue Lysate at 10 µg

Lane 4:

MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 52 kDa

Observed band size: 24 kDa,63 kDa

true

Exposure time: 1min

• WB

CiteAb

Western blot - Anti-SNRP70/U1-70K antibody (AB83306)

Western Blotting using Anti-SNRP70/U1-70K antibody, ab83306. Publication image from Pagani, F. et al., 2016, Nat Commun, 27041075. Legend direct from paper.

ExSpeU1s SM25 mutants with defective protein binding.(a) Schematic representation of RNA secondary structures and sequences of SM25 ExSpeU1 mutants. Highlighted modified nucleotides are shown in grey. (b) Expression levels, quality and nuclear distribution of the ExSpeU1 mutant RNAs. Representative northern blot analysis of ExSpeU1 mutants in total and nuclear RNA fractions. RNA was probed for ExSpeU1 SM25 and for U6 and the histograms below each northern blot show the ExSpeU1 SM25 variants' expression levels relative to U6. Values are the mean±s.e.m. of four independent experiments; *P≤0.05, **P≤0.01, ***P≤0.001. (c,d) Purity of total (T) and nuclear (N) fractions assessed by WB using Human Nuclear Matrix Protein p84, tubulin Ab and by EtBr staining of sRNAs loaded on 8% urea– polyacrylamide gel electrophoresis gel. Transfer RNA (tRNA) is missing in the nuclear fraction. (e) Affinity purification of ExSpeU1 SM25 mutants. Nuclear extracts (NE) from Hek293 cells transfected with the indicated constructs were incubated with the SM25 biotinylated oligonucleotide. Affinity-purified snRNPs were analysed by WB using antibodies against U1–70K, U1A and U1C. (f) RNA-IP analysis of SM25 ExSpeU1 variants. Hek293 NE from cells transfected with ExSpeU1 SM25 variants or not trasfected cells were incubated with antibodies against U1A, 70K, U1C and Sm proteins. RNAs purified from the RIP complexes were analysed by northern blotting. Pulled-down complexes were also analysed by northern blotting with U1 wild-type probe (Supplementary Fig. 9).

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• WB

CiteAb

Western blot - Anti-SNRP70/U1-70K antibody (AB83306)

Western Blotting using Anti-SNRP70/U1-70K antibody, ab83306. Publication image from Pagani, F. et al., 2016, Nat Commun, 27041075. Legend direct from paper.

Protein composition of ExSpeU1s and endogenous U1 snRNP.(a) Schematic representation of U1 snRNA secondary structure and associated proteins along with the RNA oligonucleotides used in affinity purification, RIP and EMSA. (b) Affinity purified CF11, SM25 and FIX9 ExSpeU1s contain 70K, U1A and U1C. Nuclear extracts (NE) from Hek293 cells, transfected with the indicated ExSpeU1s, or not transfected cells were incubated with the corresponding biotinylated 2′-O-methyl-RNA oligonucleotides. Affinity-purified snRNPs were analysed by western blotting using antibodies against U1–70K, U1A and U1C. (c) RNA-immunoprecipitation analysis of SM25 ExSpeU1. Hek293 NEs from cells transfected with ExSpeU1 SM25 or not transfected cells were incubated with antibodies against U1A, 70K and U1C. RNAs and proteins purified from the RIP complexes were analysed by northern and western blotting, respectively with the indicated probes/antibodies. Mock Ab corresponds to anti-tubulin. (d) CF11 ExSpeU1 snRNP has the same electrophoretic mobility as normal U1 in EMSA. Radiolabelled RNA oligonucleotides complementary to normal U1 (lanes 1–5) or ExSpeU1 CF11 (lanes 6–10) were incubated with nuclear extracts transfected with ExSpe CF11 or mock (not transfected cells). Addition of the indicated antibodies super-shifted the complexes in U1wt and ExSpe CF11. Control EMSA experiments are shown in Supplementary Fig. 5.

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• WB

CiteAb

Western blot - Anti-SNRP70/U1-70K antibody (AB83306)

Western Blotting using Anti-SNRP70/U1-70K antibody, ab83306. Publication image from Pagani, F. et al., 2016, Nat Commun, 27041075. Legend direct from paper.

Protein composition of ExSpeU1s and endogenous U1 snRNP.(a) Schematic representation of U1 snRNA secondary structure and associated proteins along with the RNA oligonucleotides used in affinity purification, RIP and EMSA. (b) Affinity purified CF11, SM25 and FIX9 ExSpeU1s contain 70K, U1A and U1C. Nuclear extracts (NE) from Hek293 cells, transfected with the indicated ExSpeU1s, or not transfected cells were incubated with the corresponding biotinylated 2′-O-methyl-RNA oligonucleotides. Affinity-purified snRNPs were analysed by western blotting using antibodies against U1–70K, U1A and U1C. (c) RNA-immunoprecipitation analysis of SM25 ExSpeU1. Hek293 NEs from cells transfected with ExSpeU1 SM25 or not transfected cells were incubated with antibodies against U1A, 70K and U1C. RNAs and proteins purified from the RIP complexes were analysed by northern and western blotting, respectively with the indicated probes/antibodies. Mock Ab corresponds to anti-tubulin. (d) CF11 ExSpeU1 snRNP has the same electrophoretic mobility as normal U1 in EMSA. Radiolabelled RNA oligonucleotides complementary to normal U1 (lanes 1–5) or ExSpeU1 CF11 (lanes 6–10) were incubated with nuclear extracts transfected with ExSpe CF11 or mock (not transfected cells). Addition of the indicated antibodies super-shifted the complexes in U1wt and ExSpe CF11. Control EMSA experiments are shown in Supplementary Fig. 5.

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