700129

antibody from Invitrogen Antibodies

Targeting: CREB1

Western blot (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

700129

Provider

Invitrogen Antibodies

Product name

Phospho-CREB (Ser133) Recombinant Rabbit Monoclonal Antibody (B16H16L26)

Antibody type

Monoclonal

Antigen

Other

Description

Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.

Antibody clone number

B16H16L26

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of CREB (pS133) was performed by loading 20 µg of MDA-MB-231 (lane1), MCF7 (lane2), A549 (lane3), A431 (lane4) and HEK-293 (lane5) cell lysates using Novex®NuPAGE®4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. CREB (ps133) was detected at ~43 kDa using CREB Recombinant Rabbit Monoclonal Antibody (Product # 700129) at 1-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with phosphopeptide (10 µg/mL) as shown in the corresponding blot on right. Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of CREB in NIH-3T3 cells treated with PDGF (30 µg) using a CREB recombinant rabbit monoclonal antibody (Product # 700129) at a dilution of 0.01 µg/mL. NBT/BCIP was used as the substrate (Product # WB7105). Results show a band at ~43 kDa. The second ~37 kDa band is seen in many cell types and is probably a close member of the ATF family. Competition assays show the disappearance of both the bands.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometry analysis of CREB [pS133] was done on HeLa cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª CREB [pS133] Recombinant Rabbit Monoclonal Antibody (700129, red histogram) or with rabbit isotype control (pink histogram) at 1-3 µg/million cells in 2.5% BSA. After incubation at room temperature for 2-3 hours, the cells were labeled with Alexa Fluor¨ 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.