We detected target proteins in liver cells derived from induced pluripotent stem (iPS) cells, as well as in cells treated for Protein Kinase C activation. These proteins are related to cellular differentiation and phosphorylation processes. We employed three different chemiluminescent reagents with varying sensitivities to carry out the detection.

Chemiluminescent reagent (Recommended dilution rate for secondary antibodies)	Actual dilution rate	iPS cell-derived liver cells	PKC-activated HEK293 cells PKC-activated HEK293 cells
Chemi Lumi One Ultra （1:50,000 ～ 1:500,000）	100,000x
Chemi Lumi One Super （1:10,000 ～ 1:100,000）	20,000x
Chemi Lumi One L （1:2,500 ～ 1:25,000）	5,000x

Bulue : Anti-Mouse IgG（#21860）　Pink : Anti-Rabbit IgG（#21858）

Sample:

① Control（l iPS cells 10 μg）
② iPS cell-derived liver cells 10 μg
③ Control（l HEK293 cells 10 μg）
④ PKC-activated HEK293 cells 10 μg

Primary antibody:

Diluted with Bullet ImmunoReaction Buffer (#18439-85) and incubated for 30 mins at room temperature

【αAT】	（Proteintech #66135-1-Ig）
【Oct4】	（Abcam #ab19857）
【β-Actin】	（MBL #M177-3）
【pERK】	（CST #4370S）
【ERK】	（Santa Cruz #sc-514302）
【GAPDH】	（Novus Biologicals #NB300-322）

Secondary antbody:

Diluted with Bullet ImmunoReaction Buffer (#18439-85) and incubated for 30 mins at room temperature.

Comparison of detection sensitivity by western blotting

We detected samples that were serially diluted two-fold, ranging from 16 μg/well to 20,000-fold dilution, using Anti-Mouse IgG and Anti-Rabbit IgG antibodies sourced from various companies. Our product demonstrated equal or higher sensitivity compared to products from other companies.

Sample:

HeLa Cell Suspension (diluted by 2x 16 µg/well)

SDS-PAGE:

Bullet PAGE Plus Precast Gel, 5-20%, 17wells (#21794-54)　400 V

Transfer:

Bullet Semi-dry Transfer One (#15353-01)　25 V for 10 mins.

Blotting:

Bullet Blocking One for Western Blotting (#13779) for 5 mins at RT

Primary antibody reaction:

Each product diluted by t-TBS for 60 mins at RT
[Secondary antibody; Anti-Mouse IgG] β-Actin antibody(C4)(Santa Cruz #sc-47778) diluted 2,000x
[Secondary antibody; Anti-Rabbit IgG] PCNA antibody (Novus #NB100-456)diluted 5,000x

Secondary antibody reaction:

Each product diluted by t-TBS for 60 mins at RT

Detection:

Chemi-Lumi One Super（#02230）
ChemiDoc Touch MP (Bio-Rad laboratories)(Chemiluminescence mode)
Exposure time [secondary antibody; Anti-Mouse IgG] for 2 mins. [secondary antibody; Anti-Rabbit IgG] for 3 mins.

Western Blotting by using this product

Performed immunoprecipitation (IP) on HEK293T cells transfected with FLAG-STAT5, followed by solubilization, and detected proteins using western blotting.

＜Conditions＞

Sample:

①, ⑥Samples after IP (Control; HEK293T cells) ​
②, ⑦Samples after IP（FLAG-STAT5 HEK293T cells） ​
③ ～ ⑤, ⑧, ⑨Cell lysates（FLAG-STAT5 HEK293T cells）

[Migration distance]③ 2 µL, ④ 4 µL, ⑤ 8 µL, ⑧ 3 µL, ⑨ 9 µL

IP antibody / Primary antibody:

1. ANTI-FLAG^® antibody produced in rabbit（SIGMA #F7425）​ / ANTI-FLAG^® M2 antibody produced in mouse（SIGMA #F3165）
2. ANTI-FLAG^® M2 antibody produced in mouse（SIGMA #F3165） / ANTI-FLAG^® antibody produced in rabbit（SIGMA #F7425）

Detection:

ECL Western Blotting Detection Reagents（Cytiva #RPN2209）

ANTI-FLAG^®is a registered trademark of Merck Kommanditgesellschaft auf Aktien.
ECL is a trademark of Global Life Sciences Solutions USA LLC and its affiliated companies, which operates as Cytiva.

【User evaluation】
For Anti-Mouse IgG, this product exhibited performance equally well or better than Company A's offering,with fewer non-specific bands attributed to the light chain of the immunoprecipitation antibody. Similarly, for Anti-Rabbit IgG, this product demonstrated performance equivalent to that of Company A's product.

Data courtesy of Dr. Yuichi Sekine, Lecturer. Laboratory of Cell Biology. Kyoto Pharmaceutical University