HMGB1 antibody

Catalog No. ABIN1951825

Product Details anti-HMGB1 Antibody

Target: HMGB1 (High Mobility Group Box 1 (HMGB1))

Reactivity: Human, Mouse, Cow, Pig, Mammalian

Host: Mouse

Clonality: Monoclonal

Conjugate: This HMGB1 antibody is un-conjugated

Application: Western Blotting (WB), Immunofluorescence (IF), Immunocytochemistry (ICC)

Specificity: Immunostaining Cell Cultures
1. Draw of culture medium with aspirator and add 1 ml of 3.7 % formalin in PBS solution to the dish. (make up from 10mls
Fisher 37% formalin plus 90mls PBS, the Fisher formalin contains 37% formaldehyde plus about 1% methanol which may be
relevant sometimes). Let sit at room temp for 1 minute. (can add 0.1% Tween 20 to PBS used here and all subsequent steps
to reduce background, probably best not to do this first time round though as it may extract your antigen or help wash your
cells off the dish).
2. Take off the formalin/PBS and add 1ml of cold methanol (-20°C, kept in well sealed bottle in fridge). Let sit for no more
than 1 minute.
3. Take off methanol and add 1ml of PBS, not letting the specimen dry out. To block nonspecific antibody binding can add
~10ml (=1%) of goat serum (Sigma), and can incubate for 30 minutes. Can then add antibody reagents. Typically 100ml of
hybridoma tissue culture supernatent or 1ml of mouse ascites fluid, affinity purified or crude serum. Incubate for 1 hour at
room temp. (or can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight, exact time not too critical). Can do very
gentle shaking for well adherent cell lines (3T3, Hek293 etc.).
4. Remove primary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give
three washes in PBS.
5. Add 0.5 mls of secondary antibody. These are fluorescently labeled Goat anti mouse or rabbit antibodies and are
conjugated to ALEXA dyes and are from Molecular probes (Eugene Oregon, the ALEXA dyes are sulphonated rhodamine
compounds and are much more stable to UV than FITC, TRITC, Texas red etc.). Typically make 1:2,000 dilutions of these
secondaries in PBS plus 1% goat serum, BSA or non fat milk carrier. Incubate for 1 hour at room temp. (or can go at 37°C
for 30 minutes to 1 hour, or can do 4°C overnight). Can do gentle shaking for well adherent cell lines (3T3, HEK293 etc.).
6. Remove secondary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give
three washes in PBS.
7. Drop on one drop of Fisher mounting medium onto dish and apply 22mm square coverslip. View in the microscope!

Western Blotting
1. Run gel as usual. Take gel out of electrophoresis apparatus. Cut into segments as required, Part of gel can be stained
directly in Coomassie brilliant blue R-250 (2.5 g Coomassie Brilliant Blue R-250, 450 mls methanol, 100 mls glacial acetic
acid, water to 1 liter). Part to be used for electroblotting is put into tap water on shaker, after first having marked it
unambiguously to identify top/bottom, left and right etc.
2. Leave in water on shaker for 5 minutes. This step can be substituted by washing the gel in electro-transfer buffer (see
below) for 5 minutes.
3. We use a semidry blotter, which we have found to be quicker, more economical and easier than fully submerged blotting
methods. We cut Whatman 3M filter papers to the size of our gels, and place three of these onto the semi dry blotter. These
are then wet with transfer buffer (we routinely use 3.03 g Tris base, 14.4 g Glycine, 10% Methanol per liter). The gel is put
onto the filters and a prewetted nitrocellulose filter is put ontop of the gel. Alternately put a PVDF membrane on top, if you

Purification: Aff - Purified

Immunogen: Full length recombinant human HMGB1 protein

Isotype: IgG2b

Application Details

Application Notes: Immunofluorescence: 1:1,000 or higher , Western blot: 1:1,000-2,000. Dilution listed only as recommendations. Optimal dilutions should be determined by end user.

Restrictions: For Research Use only

Handling

Format: Liquid

Concentration: 1.0 mg/mL

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: 4 °C

Storage Comment: Antibody can be aliquotted and stored frozen at -20° C to -70° C in a manual defrost freezer for six , months without detectable loss of activity. The antibody can be stored at 2° - 8° C for 1 month without , detectable loss of activity.

Target Details for HMGB1

Target: HMGB1 (High Mobility Group Box 1 (HMGB1))

Alternative Name: HMGB1 (HMGB1 Products)

Synonyms: HMG1 antibody, HMG3 antibody, SBP-1 antibody, DEF antibody, HMG-1 antibody, Hmg1 antibody, amphoterin antibody, p30 antibody, hmgb1 antibody, ik:tdsubc_1a5 antibody, wu:fb23c02 antibody, xx:tdsubc_1a5 antibody, zgc:56110 antibody, zgc:77104 antibody, hmg-1 antibody, hmg3 antibody, sbp-1 antibody, hmg1 antibody, HMGB1 antibody, Ac2-008 antibody, high mobility group box 1 antibody, high-mobility group box 1 antibody, high mobility group box 1a antibody, high mobility group box 1 L homeolog antibody, high mobility group protein B1 antibody, HMGB1 antibody, Hmgb1 antibody, hmgb1 antibody, hmgb1a antibody, hmgb1.L antibody, LOC100359149 antibody

Pathways: p53 Signaling, Regulation of Muscle Cell Differentiation, Skeletal Muscle Fiber Development, Positive Regulation of Endopeptidase Activity, Regulation of Carbohydrate Metabolic Process, Toll-Like Receptors Cascades, Smooth Muscle Cell Migration, Inflammasome