HCLS1 Mouse Monoclonal Antibody [Clone ID: 3A3]

CAT#: AM26574AF-N

HCLS1 mouse monoclonal antibody, clone 3A3, Azide Free


Product Images

Specifications

Product Data
Clone Name 3A3
Applications IP, WB
Recommended Dilution Western blot: 1 µg/ml for chemiluminescence detection system.
Immunoprecipitation: 10 µg/500µl of cell extract from 5x106 cells.
Reactivities Human, Mouse, Rat
Host Mouse
Isotype IgG1
Clonality Monoclonal
Immunogen Recombinant human HS1
Specificity This antibody reacts with HS1
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 Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing.
Stability Shelf life: One year from despatch.
Predicted Protein Size 75 kDa
Gene Name hematopoietic cell-specific Lyn substrate 1
Background The HS1 protein is one of the major substrates of non-receptor-type protein-tyrosine kinases and is phosphorylated immediately after crosslinking of the surface IgM on B cells. It is reported that HS1 protein plays a crucial role in the B-cell antigen receptor-mediated signal transduction pathway that leads to apoptosis.
Synonyms LckBP1
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 protease inhibitors at appropriate concentrations. Incubate it at 4 o C with rotating for 30 minutes; thereafter, briefly sonicate the mixture (up to 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the 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 2 minutes and centrifuge. Load 10 μ L of sample per lane on a 1-mm-thick SDS-polyacrylamide gel and carry out electrophoresis.
5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacturer's manual for precise transfer procedure.
6) To reduce nonspecific binding, soak the membrane in 5% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C.
7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1% skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on the conditions.)
8) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 3 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 off the membrane, and incubate membrane with an appr opriate chemiluminescence reagent for 1 minute.
12) Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap.
13) Expose the membrane onto an X-ray film in a dark room for 3 minutes.
14) Develop the film under usual settings. The conditions for exposure and development may vary
(Positive controls for Western blotting; Raji, Jurkat, WR19L, PC12)

Immunoprecipitation
1) Wash cells (approximately 1 x 10e7 cells) 3 times with PBS and resuspend them in 1000 μ L of cold Lysis buffer (50 mM Tris-HCl pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) containing protease inhibitors at appropriate concentrations. Incubate it at 4 o C with rotating for 30 minutes; thereafter, briefly sonicate the mixture (up to 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another fresh tube.
3) Add 50 μ L of 50% protein A agarose beads in the supernatant. Incubate it at 4 o C with rotating for 60 minutes.
4) Centrifuge the tube at 12,000 x g for 5 minutes at 4 o C. Supernatant is equally divided into another fresh two tube.
5) Add the mouse IgG1 isotype control antibody or anti-HS1 (3A3) antibody at the amount of as suggested in the APPLICATIONS to the supernatant. Vortex briefly and incubate with gently agitation for 60-120 minutes at 4 o C.
6) Add 20 μ L of 50% protein A agarose beads into the tube. Mix well and incubate with gentle agitation for 30-60 minutes at 4 o C.
7) Wash the beads 3-5 times with ice-cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds).
8) Resuspend the beads in 30 μ L of Laemmli’s sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Use 15 μ L/lane for the SDS-PAGE analysis. (See SDS-PAGE & Western blotting .)
(Positive control for Immunoprecipitation; Jurkat)

Reference Data

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