Hsp70 was immunoprecipitated using 0.5mg Rat Testis tissue lysate, 5µg of Mouse monoclonal to Hsp70 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, Rat Testis tissue lysate 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 ab47454.

Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

Band: 70kDa, non specific bands - 50kDa: We are unsure as to the identity of this extra band; Hsp70

Immunohistochemistry analysis using ab47454 (1:100) staining paraffin-embedded mouse backskin. Stained for 1 hour at RT. Secondary Antibody: FITC Goat Anti-Mouse (green) at 1:50 for 1 hour at RT.

Anti-Hsp70 antibody [N27F3-4] (ab47454) at 1/500 dilution + Apteronotus leptorhynchus brain tissue lysate at 50 µg

Secondary
Alexa Fluor® 488-conjugated Goat anti-mouse IgG polyclonal at 1/1000 dilution

Performed under reducing conditions.

Predicted band size: 70 kDa
Observed band size: 70 kDa


Exposure time: 5 minutes

See Abreview

All lanes : Anti-Hsp70 antibody [N27F3-4] (ab47454) at 1 µg/ml

Lane 1 : Cell lysate prepared from human A431 cell line
Lane 2 : Cell lysate prepared from human A549 cell line
Lane 3 : Cell lysate prepared from human HCT116 cell line
Lane 4 : Cell lysate prepared from human Hela cell line
Lane 5 : Cell lysate prepared from human HEK293 cell line
Lane 6 : Cell lysate prepared from human HepG2 cell line
Lane 7 : Cell lysate prepared from human HL-60 cell line
Lane 8 : Cell lysate prepared from human HUVEC cell line
Lane 9 : Cell lysate prepared from human Jurkat cell line
Lane 10 : Cell lysate prepared from human MCF7 cell line
Lane 11 : Cell lysate prepared from human PC3 cell line
Lane 12 : Cell lysate prepared from human T98G cell line
Lane 13 : Lysate prepared from rat brain tissue

Lysates/proteins at 10 µg per lane.

Predicted band size: 70 kDa

ICC/IF image of ab47454 stained Hek293 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab47454, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.