SAP155 (SF3B1) Mouse Monoclonal Antibody [Clone ID: 1A5]

CAT#: AM26484AF-N

SAP155 (SF3B1) mouse monoclonal antibody, clone 1A5, Azide Free


Product Images

Specifications

Product Data
Clone Name 1A5
Applications WB
Recommended Dilution

Western blot: 1 μg/ml for chemiluminescence detection system. For details see protocol below.
Not recommended for Immunohistochemistry or Immunoprecipitation.

Reactivities Hamster, Human, Mouse
Host Mouse
Isotype IgG2b
Clonality Monoclonal
Immunogen Recombinant mouse Sap155
Specificity

This antibody reacts with Sap155.

Formulation PBS containing 50% glycerol, pH 7.2. No preservative is contained.
State: Azide Free
State: Liquid Ig fraction
Concentration lot specific
Purification Protein A agarose
Conjugation Unconjugated
Storage

Store undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer.
Avoid repeated freezing and thawing.

Stability Shelf life: one year from despatch.

Gene Name splicing factor 3b subunit 1
Background

SF3 is a U2 snRNP-associated protein complex essential for spliceosome assembly and splicing catalysis of the major spliceosome. SF3 contains the Spliceosome-Associated Proteins, SAP 49, 130, 145, and 155. SAP155/Sf3b1 is an essential subunit of the U2 snRNP for mRNA splicing and has also been identified in the minor (U12-dependent) spliceosome. SAP155 interacts with the mammalian PcG (Polycomb group) proteins, Mel18 and Ring1B by the yeast two-hybrid system. SAP155 contains numerous Cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway. SAP155 serves as a substrate for cyclin E-cdk2 in vitro, suggesting that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.

Synonyms Splicing factor 3B subunit 1, SAP-155, SAP 155, SF3b155
Note This product was originally produced by MBL International.

Protocol:

SDS-PAGE & Western Blotting
1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) containing appropriate protease inhibitors. Incubate it at 4oC with rotating for 30 minutes, then sonicate briefly (up to 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4oC and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the cold Lysis buffer to make 8 mg/mL solution.
3) Mix the sample with equal volume of Laemmli’s sample buffer.
4) Boil the samples for 3 minutes and centrifuge. Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis.
5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure.
6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4oC.
7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1% skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.)
8) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 6 times).
9) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
10) Wash the membrane with PBS-T (5 minutes x 6 times).
11) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 minute.
12) Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap.
13) Expose to an X-ray film in a dark room for 5 minutes.
14) Develop the film as usual. The condition for exposure and development may vary.
(Positive controls for Western blotting; cell lines)

Reference Data

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