OxPhos Antibody Cocktail on Bone Marrow Macrophages and Osteoclasts

May 12, 2014

Internal Medicine - Bone and Mineral Diseases
Washington University in St. Louis
Graduate Research Assistant

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Company:

Abcam

Product Name:

MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail

Catalog Number:

(ab110413)

Browse Similar Products: Oxphos Western Blot Antibodies

We aim to study whether a defect in NF-kB signaling results in defect in mitochondria oxidative phosphorylation subunit expression. The goal was to use our KO mice deficient in NF-kB signaling and determine if lack of the transcription factor resulted in reduced expression of mitochondria markers at the protein level. We chose this product because it has been used in myocytes but not shown to work in bone marrow derived macrophages or osteoclasts.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Bone marrow derived macrophages (BMMs) and osteoclasts (20ug protein per lane). Make sure when denaturing in loading dye to add beta mercaptamethanol and only boil at 55deg C, or you will loose some OxPhos subunits on blot.

Primary Incubation

1:1000 dilution, overnight in 4deg diluted in 5% milk, 0.02% TBS-Tween

Blocking Agent

5% milk, 0.02% TBS-Tween

Secondary Incubation

1:2000 dilution Goat anti mouse IgG-HRP ab (santa cruz sc-2005) (0.2ug/mL final concentration)

Tertiary Incubation

Advansta WesternBright Quantum HRP substrate (K-12042-D10)

Detection

Syngene Gel Doc

Results Summary

Results show that when NF-kB factor is present (in WT), during osteoclastogenesis (stimulation with RANK ligand), oxphos subunits increase. However when NF-kB factor is knocked out, many of the OxPhos subunits are not upregulated (as evidenced by decreased expression of subunits in KO samples). This suggests that by using this antibody cocktail, we can reliably detect oxphos subunit expression in BMMs and osteoclasts from in vitro culture.

Additional Notes

Lysates resolved in a 12% gel, used semi-dry transfer for 17' at constant 15V.

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Summary

The Good

Antibody is extremely sensitive and lasts >1yr with repeated use in same diluted working stock

The Bad

Price: it is expensive

The Bottom Line

Would highly recommend using this cocktail for BMMs and osteoclast lysates to study mitochondria oxphos subunit expression.

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