Figure 1: JAK2 Target Protein Structure.

Introduction to JAK2

Protein Function

• Non-receptor tyrosine kinase involves various processes such as cell growth, development, differentiation, or histone modifications.
• Mediates essential signaling events in both innate and adaptive immunity. In the cytoplasm, it plays a pivotal role in signal transduction via its association with type I receptors such as growth hormone (GHR), prolactin (PRLR), leptin (LEPR), erythropoietin (EPOR), and thrombopoietin (THPO); or type II receptors, including IFN-alpha, IFN-beta, IFN-gamma, and multiple interleukins.
• Following ligand binding to cell surface receptors, phosphorylates specific tyrosine residues on the cytoplasmic tails of the receptor, creating docking sites for STAT proteins.

Protein Characteristics

• Auto-phosphorylation of JAK2 is crucial for regulating its activity.

Protein Expression

• JAK2 is ubiquitously expressed throughout most tissues, including blood, bone marrow, and lymph nodes.

Protein Localization

• Endomembrane system, Cytoplasm, Nucleus.

Figure 2: JAK2 ICC image, Anti-JAK2 (phospho Y1007 + Y1008) Antibody [E132] (ab32101), Anti-JAK2 Antibody [EPR108(2)] (ab108596). Green: JAK2, Red: Tubulin, Blue: DAPI.

Isoforms & post-translational modifications

• Human (O60674): 130 kDa (predicted)
• Mouse (Q62120): 130 kDa (predicted)
• Rat (Q62689): 130 kDa (predicted)
• Multiple phosphorylation sites.

WB Experiment Tips

Precautions

• Unstimulated samples (e.g., cells or brain tissue) may make detecting phosphorylated JAK2 protein (Y1007 + Y1008) challenging. We recommend using pervanadate for pre-stimulation.
• Use appropriate positive controls depending on the form of JAK2 protein being detected.
• The predicted molecular weight of the protein is approximately 130 kDa, so please follow specific guidelines for handling large proteins during electrophoresis and transfer processes.

Positive Controls

• JAK2: Cell lysates from K562, Jurkat, A549, C6, and similar cells.
• JAK2 (phospho Y1007 + Y1008): Cell lysates from Jurkat or C6 cells treated with 50 mM Pervanadate for 5 minutes.

Negative Control

• Human JAK2 knockout A549 cell lysate (ab256963)

Example Results

Figure 3: WB - Anti-JAK2 antibody [EPR108(2)] (ab108596)

Lane 1: 20 µg A549 cell lysate
Lane 2: 20 µg JAK2 knockout A549 cell lysate
Lane 3: 20 µg K562 cell lysate
Lane 4: 20 µg Daudi cell lysate

Primary Antibodies: Anti-JAK2 antibody [EPR108(2)] (ab108596) at 1/1000 dilution
Anti-GAPDH antibody [6C5] (ab8245) at 1/20000 dilution
Secondary Antibodies: Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution.

Result Description: JAK2 (green), GAPDH (red)
Predicted Band Size: 131 kDa
Observed Band Size: 131 kDa

Figure 4: WB - JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)

Lane 1: 10 µg Untreated Jurkat cells whole cell lysates
Lane 2: 10 µg Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates
Lane 3: 10 µg Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates. Then the membrane was incubated with Alkaline phosphatase.

Primary Antibody: JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101) at 1/5000 dilution
Secondary Antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted Band Size: 130 kDa
Observed Band Size: 120 kDa

Figure 5: WB - JAK2 (phospho Y1007 + Y1008) Antibody [E132] (ab32101)

Lane 1: 15 µg Mouse hippocampus lysate
Lane 2: 15 µg Mouse P240 hippocampus lysate
Lane 3: 15 µg Mouse P7 hippocampus lysate
Lane 4: 15 µg Rat hippocampus lysate
Lane 5: 15 µg Rat P7 hippocampus lysate
Lane 6: 15 µg Rat cerebral cortex lysate
Lane 7: 15 µg Human brain lysate
Lane 8: 15 µg Mouse brain lysate
Lane 9: 15 µg Rat brain lysate
Lane 10: 15 µg C6 (rat glial tumor glial cells) whole cell lysate
Lane 11: 15 µg C6 treated with 50 mM Pervanadate for 5 minutes whole cell lysate

Primary Antibody: JAK2 (phospho Y1007 + Y1008) Antibody [E132] (ab32101)
Secondary Antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted Band Size: 130 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 a protease inhibitor cocktail to prevent degradation of target proteins.
2. Add a phosphatase inhibitor cocktail to prevent dephosphorylation during extraction.
3. Keep samples on ice throughout the entire sample preparation process.
4. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.
5. It is recommended to use positive control.

Electrophoresis:

1. For target proteins with larger molecular weights (e.g., >100 kDa), we recommend using an 8% separating gel for electrophoresis.

Transfer:

1. For target proteins with a higher molecular weight, it is recommended that SDS be added to the transfer buffer at a final concentration of 0.1%.
2. For target proteins with a higher molecular weight, we advise using a PVDF membrane with a pore size of 0.45 μm.
3. We recommend using 10% methanol or lower concentration in the transfer buffer for target proteins with a higher molecular weight.
4. We recommend staining the membrane with Ponceau S after the transfer to confirm its success.

References

1. M Sakatsume, K Igarashi, K D Winestock, G Garotta, A C Larner, D S Finbloom. The Jak kinases differentially associate with the alpha and beta (accessory factor) chains of the interferon gamma receptor to form a functional receptor unit capable of activating STAT transcription factors. J Biol Chem. 1995 Jul 21;270(29):17528-34. doi: 10.1074/jbc.270.29.17528.
2. A Saltzman, M Stone, C Franks, G Searfoss, R Munro, M Jaye, Y Ivashchenko. Cloning and characterization of human Jak-2 kinase: high mRNA expression in immune cells and muscle tissue. Biochem Biophys Res Commun. 1998 May 29;246(3):627-33. doi: 10.1006/bbrc.1998.8685.
3. Daniel C Berry, Hui Jin, Avijit Majumdar, Noa Noy. Signaling by vitamin A and retinol-binding protein regulates gene expression to inhibit insulin responses. Proc Natl Acad Sci U S A. 2011 Mar 15;108(11):4340-5. doi: 10.1073/pnas.1011115108. Epub 2011 Feb 23.