Reliable Antibody - But Sometimes Gives Two Bands

Cell Biology
Albert Einstein College of Medicine
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Cell Signaling Technology

Product Name:

Phospho-Akt (Ser473) (D9E) XP Rabbit mAb

Catalog Number:

4060

The general aim of my research is to study signaling pathways involved in the pathogenesis of leukemia by specifically studying signaling in acute myeloid leukemia (AML) stem cells. The specific goal of the experiment for which I used the product was to determine whether AML cells have active/phosphorylated Akt. The reason I selected this product is because I have had previous success with Cell Signaling phospho-antibodies.

Experimental Design and Results Summary

Applications

Western Blot

Sample

HEL AML cell line

Primary Incubation

1:1000, overnight at 4 degrees C with rocking

Blocking Agent

TBS + 0.1% Tween + 5% w/v BSA

Secondary Incubation

1:5000 anti-rabbit-HRP, 1 hour at room temperature with rocking

Tertiary Incubation

None

Detection

ECL

Results Summary

Clear band at the expected size (60 kDa) but also a band between 55-60 kDa and ~90 kDa.

DOI or PMID #

N/A

Additional Notes

N/A

Related Categories

Image Gallery

Summary

The Good

Responds to cytokine stimulation

The Bad

Unclear whether the antibody binds to other proteins aside from Akt as there are extra bands

The Bottom Line

Recommended

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