All lanes : Anti-Lactate Dehydrogenase antibody (ab47010)

Lane 1 : LDHA recombinant
Lane 2 : LDHB recombinant
Lane 3 : LDHC recombinant
Lane 4 : HeLa

Predicted band size: 37 kDa


Exposure time: 30 seconds

Observed bands

Lane 1 :40 kDa

Lane 2:37 kDa

Lane 3:62 kDa

Lane 4: 37 kDa

IHC image of Lactate Dehydrogenase staining in human breast carcinoma FFPE section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab47010, 1µg/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.

ICC/IF image of ab47010 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab47010, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

LDHA was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to LDHA and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab47010.

Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band: 36kDa; LDHA

All lanes : LDHA antibody (ab47010) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 4 : HEK293 Human embryonic kidney cell line Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 37 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

ab47010 at a 1/400 dilution staining LDHA in human skeletal muscle sections by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections) incubated for 20 minutes at 25°C. Heat mediated antigen retrieval used, EDTA ph 9.0, 100°C for 20 minutes. Secondary used undiluted, goat anti-mouse/rabbit IgG conjugated to HRP polymer.

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Anti-Lactate Dehydrogenase antibody (ab47010) at 1/500 dilution + Zebrafish whole cell lysate at 10 µg

Secondary
HRP conjugated Goat anti-rabbit monoclonal secondary antibody at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 37 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?
Additional bands at: 22 kDa (possible non-specific binding)


Exposure time: 30 seconds

Blocking step: 5% Milk for 1 hour at 24ºC

Primary antibody incubation for 12 hours at 4ºC

See Abreview

Anti-Lactate Dehydrogenase antibody (ab47010) at 1 µg/ml + Recombinant human LDHA protein (Active) (ab93699) at 0.1 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 37 kDa


Exposure time: 10 seconds