All lanes : Anti-Caveolin-1 antibody - Caveolae Marker (ab18199) at 1 µg/ml

Lane 1 : Wild-type A431 cell lysate
Lane 2 : A431 knockout CAV1 cell lysate
Lane 3 : A549 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 21-24 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab18199 observed at 21-24 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab18199 was shown to react with Caveolin-1 in wild-type A431 cells in western blot. Loss of signal was observed when CAV1 knockout sample was used. Wild-type and CAV1 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab18199 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Caveolin-1 antibody - Caveolae Marker (ab18199) at 1 µg/ml

Lane 1 : Mouse Heart Lysate
Lane 2 : Human Heart Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Alexa Fluor Goat polyclonal to Rabbit IgG (700) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 20 kDa

All lanes : Anti-Caveolin-1 antibody - Caveolae Marker (ab18199) at 1 µg/ml

Lane 1 : NIH/3T3 whole cell lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : Heart (Mouse) Tissue Lysate
Lane 4 : Heart (Rat) Tissue 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: 20 kDa
Observed band size: 20 kDa

Caveolin 1 - Caveolae Marker was immunoprecipitated using 0.5mg Mouse Heart whole tissue lysate, 5µg of Rabbit polyclonal to Caveolin 1 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, Mouse Heart whole 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^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18199.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 20kDa: Caveolin 1 - Caveolae Marker.

Anti-Caveolin-1 antibody - Caveolae Marker (ab18199) at 1 µg/ml + Recombinant Human Caveolin-1 protein (ab114170) at 0.01 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: 20 kDa


Exposure time: 1 minute

ab18199 recognizes the full length tagged recombinant Caveolin 1 protein (ab114170) which has an expected molecular weight of 46 kDa.