Figure 1: Structure of the Iba1 protein.

Iba1 Introduction

Protein function

• Iba1 (Ionized calcium-binding adapter molecule 1), also known as Allograft Inflammatory Factor 1 (AIF1), is an evolutionarily conserved cytoplasmic calcium-binding protein.
• Iba1 is a cytoskeletal binding protein that enhances membrane ruffling and RAC activation, playing a role in the RAC signaling pathway and in phagocytosis.
• Iba1 promotes proliferation of vascular smooth muscle cells and T lymphocytes. Enhances lymphocyte migration. Plays a role in vascular inflammation.
• Iba1 acts as an F-actin binding protein and also plays a role in remodeling the actin cytoskeleton. It participates in morphological changes associated with activated microglia/macrophages, widely used as a marker for microglia/macrophages in the brain and other tissues.

Protein expression

• Expressed in microglia, T-lymphocytes and peripheral blood monocytes cells.

Protein localization

• Iba1 is primarily localized to the cell membrane and cytoskeleton.

Figure 2: Iba1 ICC Experiment Results, Anti-Iba1 antibody [EPR16589] (ab178847). Green: Iba1, Red: Tubulin, Blue: DAPI.

Isoforms and post-translational modifications

• Human (P55008): Isoforms 1-3: 10-17 kDa (predicted)
• Mouse (O70200): 17 kDa (predicted)
• Rat (P55009): 17 kDa (predicted)
• Acetylation and Phosphorylation

WB Experiment Tips

Precautions

• Iba1 is a small protein with a molecular weight of approximately 17 kDa. It is recommended to use 15% SDS-PAGE gels or gradient gels for electrophoresis in WB experiments to achieve optimal results.
• Iba1 exists in three isoforms with molecular weights ranging from 10 to 17 kDa. Multiple bands may be observed in WB experiments due to these isoforms.
• For various Iba1 antibodies such as ab5076, ab153696, ab178846, and ab48004, Abcam recommends consulting the datasheet for specific protocols. Blocking with 5% non-fat milk is recommended to reduce background and obtain cleaner bands.
• Iba1 is a protein with tissue-specific expression. Some samples may exhibit weak or no expression (as depicted in Figure 4). It is crucial to select appropriate experimental samples and use positive controls to ensure the experimental system is functioning correctly.
• Iba1 protein is relatively less abundant in the brain but more in the spleen (PMID: 8912632, PMID: 29232670). It is advised to load more brain lysate or use higher concentrations of antibody to detect signals effectively in brain-related lysates.

Positive controls

• THP-1, U937, RAW 264.7 cells whole cell lysates
• human, mouse, and rat spleen lysates
• Mouse hippocampal tissue lysates
• Recombinant human Iba1 protein (ab105593)

Negative controls (low expression or no expression)

• Neurons and astrocytes in the brain
• Skeletal muscle

Example results

Figure 3: Western blot - Anti-Iba1 antibody [EPR16588] (ab178846).

Lane 1: THP-1 whole cell lysate
Lane 2: U937 whole cell lysate
Lane 3: Human spleen whole cell lysate

Predicted band size: 17 kDa
Detected band size: 15 kDa

Figure 4: Western blot - Anti-Iba1 antibody [EPR16588] (ab178846).

Lane 1 : Human Iba1 recombinant protein at 0.1 µg
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg
Lane 3 : A431 (human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 4 : NIH/3T3 (mouse embyro fibroblast cell line) whole cell lysate at 30 µg
Lane 5 : Human spleen tissue lysate at 20 µg
Lane 6 : Mouse spleen tissue lysate at 30 µg
Lane 7 : Rat spleen tissue lysate at 30 µg
Lane 8 : U937 (human histiocytic lymphoma cell line) whole cell lysate at 30 µg
Lane 9 : MOLT-4 (human lymphoblastic leukemia cell line) whole cell lysate at 20 µg
Lane 10 : THP-1 (human monocytic leukemia cell line) whole cell lysate at 30 µg
Lane 11 : THP-1 whole cell lysate, PMA treated at 30 µg
Lane 12 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 30 µg
Lane 13 : C6 (rat glial tumor cell line) whole cell lysate at 30 µg
Lane 14 : NR8383 whole cell lysate at 30 µgPredicted band size: 17 kDa

* Abcam recommends blocking in milk for cleaner blots with reduced background, in comparison to BSA.

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. It is recommended to use a 15% separating gel for electrophoresis.
2. Load at least 20μg of total protein from cell lysate or tissue homogenate.

Transfer:

1. It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.
2. For target proteins with a lower molecular weight, it is recommended to use a PVDF membrane with a pore size of 0.22μm, the transfer time should not be too long to prevent the absence of the band.
3. It is recommended not to cut the membrane and keep the entire membrane or at least 30 kDa for antibody incubation.

IHC experiment tips

• Iba1 is a calcium-binding protein specific to activated microglia or macrophages, where it interacts with cytoskeletal proteins to facilitate cell movement.
• When the host species of the primary antibody matches the species of the sample being tested, optimization of experimental conditions is necessary. For instance, when using a mouse primary antibody (ab283319) to detect mouse samples, it is recommended to block endogenous IgG binding with an appropriate IgG blocking agent after standard serum blocking to reduce non-specific staining. Additionally, using a signal amplification system such as Polymer-linked HRP secondary antibodies is advised to enhance experimental signals.
• Ensure that samples are not over-fixed in IHC experiments.

Positive controls

• Human cerebral cortex, hippocampal tissue, spleen tissue, amygdala tissue
• Rat and mouse brain tissue

Example results

Figure 5: IHC - Anti-Iba1 antibody [EPR16588] (ab178846).

Sample: Human normal hippocampus, formalin-fixed paraffin-embedded tissue section
Antigen retrieval solution: pH 6.0 sodium citrate buffer
Detection method: DAB
Other details: Experiment conducted

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 fixation:

1. The optimal fixation time for samples depends on the size and type of tissue, but for most samples, it is advisable to fix with 4% PFA at room temperature for 18-24 hours.

Blocking:

1. When conducting experiments using secondary antibodies conjugated with fluorescent labels, it is recommended to add 1% BSA and a final concentration of 0.3M glycine to the blocking solution to quench the autofluorescence induced by aldehyde groups.
2. If using HRP conjugate for detection, treat the sections with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity.

Antigen retrieval:

1. When performing immunohistochemistry experiments on paraffin sections, please choose the appropriate antigen retrieval solution according to the datasheet. We recommend using a pressure cooker for heat-induced antigen retrieval. You can try to repair the sections at 110℃ for 15 minutes.

Antibody incubation:

1. When conducting initial experiments, please select the appropriate working antibody concentration according to the antibody datasheet.
   It is recommended to use Polymer-conjugated HRP secondary antibodies to amplify the signal.

ICC experiment tips

Precautions

• Iba1 is a protein with tissue-specific expression, found in microglia, T lymphocytes, and peripheral blood monocytes. Select appropriate experimental samples and use positive controls to confirm the experimental system is functioning correctly.

Positive controls

• THP-1 cell line
• Jurkat cell line

Negative controls (low or no expression)

Neurons and astrocytes in the brain

Example results

Figure 6: Recombinant Anti-Iba1 antibody [EPR6136(2)] (ab178680)

Sample: Jurkat (human T-cell leukemia T lymphocyte) cells
Experimental Method: Fixed in 4% PFA, permeabilized with 0.1% Triton X-100
Experimental Results: Green: Iba1, Red: Tubulin, Blue: DAPI

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 fixation:

1. If using aldehydes (such as 4% PFA) for fixation, it is recommended to fix at room temperature for 10 to 20 minutes. If using organic solvents (such as methanol) for fixation, it is recommended to fix at -20°C for 5 to 10 minutes.

Permeabilization:

1. If using aldehydes (such as 4% PFA) for cell fixation, it is recommended to permeabilize with 0.1-0.25% Triton X-100 at room temperature for 10 minutes. Permeabilization is particularly critical if the target protein is located intracellularly.

Blocking:

1. It is recommended to add 10% serum from the species of the secondary antibody + 1% BSA + a final concentration of 0.3M glycine in the blocking solution to quench the autofluorescence caused by aldehyde groups.

Antibody incubation:

1. Please refer to the specific antibody's instructions to choose the appropriate working concentration of the antibody. A too high concentration of the primary antibody may cause cross-reactivity in negative cell lines, resulting in non-specific signals being detected.

References

1. D Ito, Y Imai, K Ohsawa, K Nakajima, Y Fukuuchi, S Kohsaka. Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res. 1998 Jun 1;57(1):1-9. doi: 10.1016/s0169-328x(98)00040-0.
2. Jörg O Schulze, Claudia Quedenau, Yvette Roske, Thomas Adam, Herwig Schüler, Joachim Behlke, Andrew P Turnbull, Volker Sievert, Christoph Scheich, Uwe Mueller, Udo Heinemann, Konrad Büssow. Structural and functional characterization of human Iba proteins. FEBS J. 2008 Sep;275(18) 10.1111/j.1742-4658.2008.06605.x. Epub 2008 Aug 11.