Rabbit Loading Control Antibody Panel (ab199712)

All lanes : Anti-GAPDH antibody [EPR16891] (ab181602) at 1/50000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 2 : Xenopus(X. tropicalis) muscle lysates
Lane 3 : UMNSAH/DF-1 (Transformed chicken embyronic fibroblast cells) whole cell lysates
Lane 4 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size : 36 kDa
Observed band size : 36 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-alpha Tubulin antibody [EPR13478(B)] - Loading Control (ab176560) at 1/1000 dilution (Unpurified)

Lane 1 : HeLa cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : A431 cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 10 µg per lane.

Developed using the ECL technique

Predicted band size : 50 kDa

Secondary antibodies - anti-rabbit HRP (ab6721)

All lanes : Anti-COX IV antibody [EPR9442(ABC)] - Mitochondrial Loading Control (ab202554) at 1/10000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Rat heart lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size : 20 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)


Exposure time : 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Histone H3 antibody [EPR16987] - Nuclear Loading Control and ChIP Grade (ab176842) at 1 µg

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg
Lane 3 : Drosophila embryo nuclear extract (from melanogaster embryos 0-12hr) at 10 µg
Lane 4 : S.cerevisiae (Y190) Whole Cell Lysate at 20 µg
Lane 5 : S.pombe Whole Cell Lysate at 20 µg

Secondary
Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution
Developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 15 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)


Exposure time : 5 seconds

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab176842 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes : Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading Control (ab76020) at 1/100000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 3 : 293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 4 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

Predicted band size : 113 kDa
Observed band size : 100 kDa (why is the actual band size different from the predicted?)


Exposure time : 2 minutes

Blocking and diluting buffer: 5% NFDM/TBST.