EV Antibody Database

Western Blot Antibody

Target protein:

CD146-(MelCAM)

Primary antibody:

R&D Systems MAB932 (Mouse)

No notes

Source material:

Tissue: Placental explants

Placental explant cultured with DKSFM media + 2% exo-free FBS at 37°C, 5% CO2 for 8 hours. The culture supernatant was centrifuged at 2,000 xg for 10 min at room temperature. Then, the supernatant was aliquoted and kept at -80°C until analysis.

Secondary antibody:

Sigma-Aldrich 12-349 (Goat)

The SNAP i.d.® 2.0 system was used for this incubation (10 min)

Lysis Conditions:

RIPA buffer
at
4°C
for
15 min

Gel Conditions

Minigel, Any kD % acrylamide

 

Gel type:

BioRad 4568125

Run conditions:

200 V / Variable Amps for 30 min

Protein loading:

30 𝜇g

Reducing conditions:

Dithiothreitol

No notes

Transfer Conditions

Membrane type:

PVDF

Transfer method:

Semi-dry

Transfer buffer:

Tris-glycine with 20% methanol

Transfer voltage:

25 V

Transfer current:

1.0 Amps

Transfer temperature:

22°C

Transfer duration:

0.5 hr

Blocking

Blocking buffer:

0.5% milk in TBST (0 min). The blocking, antibodies incubations and washings were done using the SNAP i.d.® 2.0 system.

Blocking temperature:

22°C

Duration:

0 hr

Primary Incubation

Antibody 1 dilution

1:500

Incubation temperature

22°C

Incubation duration

0.16 hr

Number of washes

4

Wash buffer

TBST, 0.1% Tween-20

Wash duration

0 min

Secondary Incubation

Antibody 2 dilution

1:20,000

Incubation temperature

22°C

Incubation duration

0.16 hr

Number of washes

4

Wash buffer

TBST, 0.1% Tween-20

Wash duration

0 min

Detection

Detection method

Enhanced chemiluminescence (ECL)

Expected band size

110 kDa

Result:

Strong single band

Background:

Multiple non-specific bands

Related Publications

–––

Multiple non-specific bands only in the placental lysate.

Record 10005 author: Luiza Perucci

Author Information

Email:lperucci@health.ucsd.edu
Lab Head:
Institution:
Website:
Last edit: Tue Mar 19, 2024