Figure 1: Crystal structure of the CRTC2(SeMet)-CREB-CRE complex.

CREB target introduction

Protein function

• Cyclic AMP-responsive element-binding protein 1 (CREB1/CREB) is a phosphorylation-dependent transcription factor that stimulates transcription when bound to DNA cAMP response elements (CRE). CRE is a sequence present in many viral and cellular promoters.
• It involves various cellular processes, including circadian rhythm synchronization and adipocyte differentiation.

Protein localization

• It is localized in the cell nucleus.

Figure 2: CREB experimental results image, Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 647 Anti-CREB antibody [E306] (ab194312). Red: CREB; Green: alpha Tubulin; Blue: DAPI.

Isoforms & Post-translational modifications

Human (P16220): Isoform 1-2, 35-36 kDa, Isoform 3, 25 kDa (predicted)
Mouse (Q01147): Isoform 1-2, 35-36 kDa (predicted)
Rat (P15337): Isoform 1-2, 35-36 kDa (predicted)
Multiple phosphorylation modification sites are present.

WB experiment tips

Precautions

• The predicted molecular weight of CREB is 36 kDa, and it has multiple phosphorylation sites. Phosphorylated forms of CREB need to be induced by stimulation to be detected, and the actual detected molecular weight is slightly larger than 36 kDa. Please choose appropriate stimulation induction conditions based on literature or instructions.
• When detecting phosphorylated CREB, please first confirm the content of total CREB protein in the cells.
• Phosphatase is an enzyme that can dephosphorylate the corresponding substrate. If you are unsure whether the detected protein band is the target protein with phosphorylation modification, you can use phosphatase to treat the membrane.

Positive control

• CREB (phospho S133): HeLa, NIH/3T3, C6 whole cell lysate treated with Calyculin A for 30 minutes.

Example of results

Figure 3: Anti-CREB (phospho S133) antibody [E113] (ab32096).

Lane 1: 15 µg 250ng/ml puromycin-treated HeLa whole cell lysate for 30 minutes.
Lane 2: 15 µg mouse brain tissue lysate.
Lane 3: 15 µg rat brain tissue lysate.
Lane 4: 15 µg rat cerebellum tissue lysate.
Lane 5: 15 µg mouse hippocampus tissue lysate.
Lane 6: 15 µg mouse hippocampus tissue lysate.
Lane 7: 15 µg rat cerebral cortex tissue lysate.

Predicted band size: 37 kDa.
Other: No signal detected in mouse and rat brain-related tissues.

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex protease inhibitor to prevent degradation of the target protein.
2. Add a complex phosphatase inhibitor to prevent protein dephosphorylation during extraction.
3. Sonicate the cells to enrich the target protein.
4. Keep the sample on ice throughout the sample preparation process.
5. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.

Transfer:

1. We recommend using Ponceau S staining after transfer to confirm the success of the transfer.

References

1. Katrina M Comerford, Martin O Leonard, Jorn Karhausen, Robyn Carey, Sean P Colgan, Cormac T Taylor. Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia. Proc Natl Acad Sci U S A. 2003 Feb 4;100(3):986-91. doi: 10.1073/pnas.0337412100. Epub 2003 Jan 27.
2. K Du, M Montminy. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem. 1998 Dec 4;273(49):32377-9. doi: 10.1074/jbc.273.49.32377.