Figure 1: JAK1 protein structure.

Introduction to JAK1

Protein Function

• Tyrosine kinase of the non-receptor type, involved in the IFN-alpha/beta/gamma signal pathway.
• Kinase partner for the interleukin (IL)-2 receptor as well as interleukin (IL)-10 receptor.
• Kinase partner for the type I interferon receptor IFNAR2. In response to interferon-binding to IFNAR1-IFNAR2 heterodimer, phosphorylates and activates its binding partner IFNAR2, creating docking sites for STAT proteins.
• Directly phosphorylates STAT proteins and activates STAT signaling by transactivating other JAK kinases associated with signaling receptors.

Protein Expression

• Expressed at higher levels in primary colon tumors than normal colon tissue. The expression level in metastatic colon tumors is comparable to that in normal colon tissue.

Protein Localization

• Endomembrane system.

Figure 2: JAK1 ICC image, Immunocytochemistry/Immunofluorescence - Anti-JAK1 Antibody (ab125051). Green: JAK1; Red: Tubulin; Blue: DAPI.

Isoforms & Post-translational Modifications

• Human (P23458): 133.3 kDa (predicted)
• Mouse (P52332): 133.4 kDa (predicted)
• There are multiple phosphorylation sites.
• Ubiquitinated by RNF125; leading to its degradation by the proteasome.

WB Experiment Tips

Precautions

• JAK1 can be ubiquitinated, leading to its proteasomal degradation. We recommend adding proteasome inhibitors to inhibit this degradation process and enhance detection signals.
• Phosphorylated forms of JAK1 (p-JAK1) protein may require induction by stimulation for detection. Refer to product datasheets or literature for appropriate stimulation conditions.
• When detecting p-JAK1 protein, it is advisable to simultaneously measure the total amount of JAK1 protein to ensure sufficient levels of the target protein in the cells.

Positive Controls

• JAK1: A431 whole cell lysate, NIH/3T3 whole cell lysate
• JAK1 (phospho Y1034 + Y1035): Ramos treated with 1 mM pervanadate for 30 minutes whole cell lysate

Example Results

Figure 3: Anti-JAK1 antibody [EPR349(N)] (ab133666)

Lane 1: 40 µg HAP1 whole cell lysate
Lane 2: Empty
Lane 3: 40 µg JAK1 (KO) HAP1 whole cell lysate
Lane 4: 20 µg A431 whole cell lysate

Predicted band size: 133 kDa
Observed band size: 133 kDa
Description: Green - JAK1, Red - GAPDH

Figure 4: Anti-JAK1 (phospho Y1034 + Y1035) antibody [EPR1899(2)] (ab138005)

Lane 1: 10 µg Ramos whole cell lysate
Lane 2: 10 µg Ramos treated with 1 mM pervanadate for 30 minutes whole cell lysate

Predicted band size: 133 kDa
Observed band size: 133 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. Keep samples on ice throughout the entire sample preparation process.
3. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. For target proteins with larger molecular weights (e.g., >100 kDa), we recommend using an 8% separating gel for electrophoresis.
2. Load at least 20 μg of total protein from cell lysate or tissue homogenate.

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 Müller, J Briscoe, etc. The protein tyrosine kinase JAK1 complements defects in interferon-alpha/beta and -gamma signal transduction. Nature. 1993 Nov 11;366(6451):129-35. doi: 10.1038/366129a0.
2. M Sakatsume, K Igarashi, etc. 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.
3. Kate L Del Bel, Robert J Ragotte, etc. JAK1 gain-of-function causes an autosomal dominant immune dysregulatory and hypereosinophilic syndrome. J Allergy Clin Immunol. 2017 Jun;139(6):2016-2020.e5. doi: 10.1016/j.jaci.2016.12.957. Epub 2017 Jan 19.
4. Conor N Gruber, Jorg J A Calis, etc. Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function. Immunity. 2020 Sep 15;53(3):672-684.e11. doi: 10.1016/j.immuni.2020.07.006. Epub 2020 Aug 3.
5. Judith Staerk, Anders Kallin, etc. JAK1 and Tyk2 activation by the homologous polycythemia vera JAK2 V617F mutation: cross-talk with IGF1 receptor. J Biol Chem. 2005 Dec 23;280(51):41893-9. doi: 10.1074/jbc.C500358200. Epub 2005 Oct 19.
6. Paul D Simoncic, Ailsa Lee-Loy, etc. The T cell protein tyrosine phosphatase is a negative regulator of janus family kinases 1 and 3. Curr Biol. 2002 Mar 19;12(6):446-53. doi: 10.1016/s0960-9822(02)00697-8.
7. Anna Usacheva, Sergei Kotenko, etc. Two distinct domains within the N-terminal region of Janus kinase 1 interact with cytokine receptors. J Immunol. 2002 Aug 1;169(3):1302-8. doi: 10.4049/jimmunol.169.3.1302.
8. D Novick, B Cohen, N Tal, M Rubinstein. Soluble and membrane-anchored forms of the human IFN-alpha/beta receptor. J Leukoc Biol. 1995 May;57(5):712-8. doi: 10.1002/jlb.57.5.712.