Image 1: BDNF protein structure.

BDNF Target Introduction

Protein Function

• BDNF is one of the members of the neurotrophin protein family, and it is known to affect the growth, development, differentiation, and survival of neurons.
• BDNF is a major regulatory factor in adult synaptic transmission and plasticity.
• During neural development, BDNF promotes the survival and differentiation of specific groups of neurons in the peripheral and central nervous systems, and is involved in the regulation of axon growth, development, and dendritic cell growth and morphology.
• The precursor proBDNF and mature mBDNF have opposite effects on long-term potentiation and long-term depression.

Protein Expression

• BDNF is highly expressed in brain tissues such as the hippocampus, amygdala, cerebral cortex, and cerebellum.
• BDNF is also expressed in the heart, lungs, skeletal muscles, testes, prostate, and placenta.

Protein Localization

• Secreted protein.

Image 2: ICC experimental result image of BDNF target, using the Anti-BDNF antibody [EPR1292] (ab108319). Green: BDNF, Red: Tubulin, Blue: DAPI.

Isoforms & post-translational modifications

• Human (P23560): Isoforms 1-5: 28-37 kDa (predicted)
• Mouse (P21237): Isoforms 1-2: 28-29 kDa (predicted)
• Rat (P23363): 28 kDa (predicted)
• The precursor is N-glycosylated, and glycosaminoglycan sulfated.
• Converted to mature BDNF by plasminogen (PLG).

WB experiment tips

Precautions

• Due to the existence of different isoforms and post-translational modifications of BDNF, as well as mature and immature forms of BDNF, multiple bands may appear in the WB experiment.

• The forms and levels of BDNF protein may vary in different samples, and it is reasonable to observe band sizes of 15-45 kDa.

  • ~15 kDa may be the monomeric mature form (processed) of BDNF
  • ~28 kDa may be the dimeric mature form (processed) of BDNF
  • ~35 kDa, ~45 kDa may be the immature form (unprocessed/propeptide) of BDNF

Positive control

• Recombinant human/mouse/rat BDNF protein (ab9794)
• Lysates of human, rat, and mouse brain, hippocampus, and cerebellum tissues

Example of results

Figure 3: WB-Anti-BDNF antibody [EPR1292] (ab108319).

Primary antibody: Used ab108319 at a concentration of 1/1000.
Blocking: Used Licor blocking solution.

Lane 1: Human hippocampal tissue lysate
Lane 2: Rat hippocampal lysate
Lane 3: Mouse hippocampal lysate

Predicted band size: 15 kDa

Figure 4: WB-Anti-BDNF antibody [EPR1292] (ab108319).

Primary antibody: Used ab108319 at a concentration of 1/1000.

Lane 1: Human brain tissue lysate.
Lane 2: Mouse brain tissue lysate.
Lane 3: Rat brain tissue lysate.
Lane 4: Human hippocampus tissue lysate.
Lane 5: Rat hippocampus tissue lysate.
Lane 6: Mouse hippocampus tissue lysate.
Lane 7: Human cerebellum tissue lysate.
Lane 8: Mouse cerebellum tissue lysate.
Lane 9: Rat cerebellum tissue lysate.

Predicted band size: 15 kDa.
Detected band size: 15-45 kDa.

Key control points

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

Sample preparation:

1. Add enough protease inhibitor to avoid degradation of the target protein.
2. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. For BDNF, please use high concentration (15%) separation gel or gradient gel for electrophoresis.

Transfer:

1. We strongly recommend using Coomassie Brilliant Blue staining after transfer to determine if the transfer is successful.

Antibody incubation:

1. We recommend using fresh antibodies and avoid reuse.

References

1. Michael F Egan, Masami Kojima, Joseph H Callicott, Terry E Goldberg, Bhaskar S Kolachana, Alessandro Bertolino, Eugene Zaitsev, Bert Gold, David Goldman, Michael Dean, Bai Lu, Daniel R Weinberger. The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell. 2003 Jan 24;112(2):257-69. doi: 10.1016/s0092-8674(03)00035-7.
2. S J Mowla, H F Farhadi, S Pareek, J K Atwal, S J Morris, N G Seidah, R A Murphy. Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor. J Biol Chem. 2001 Apr 20;276(16):12660-6. doi: 10.1074/jbc.M008104200. Epub 2001 Jan 10.