Nuclear Signal: Anti-BRAF35 IF

May 13, 2014

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Biological Science
Florida State University
Assistant Professor

Company:

EMD Millipore

Product Name:

Anti-BRAF35 Antibody, clone 4.21

Catalog Number:

05-641

The purpose of this experiment was to identify an antibody that was suitable for detection of BRAF35 in human cells by indirect immunofluorescence.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Human hTERT-RPE1 cells

Primary Incubation

1:100 at room temperature for 60 minutes. Staining was performed in 1 x PBS and 0.1% Tween 20

Blocking Agent

Slide was pre blocked with 3% BSA in 1 x PBS and 0.1% Tween 20

Secondary Incubation

1:100 dilution of Rhodamine Goat anti-mouse secondary diluted in 1 x PBS and 0.1% Tween-20 for 60 minutes at room temperature

Tertiary Incubation

No tertiary incubation was performed

Detection

Direct labeled goat anti-mouse antibodies

Results Summary

This antibody gives the expected nuclear distribution. The signal was moderate to weak, which may reflect protein levels in hTERT-RPE1 cells. Some cells showed stronger signals than others, which may be due to the cell cycle.

Additional Notes

Cells were grown directly on slides and washed briefly with 1 x PBS before fixing and extracting for 10 minutes at room temperature in 4% formaldehyde, 1 x PBS and 0.1% Triton-X100

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Summary

The Good

The signal was nuclear as expected with low background.

The Bad

The signal was moderate to weak, but this could be due to protein levels.

The Bottom Line

The antibody is suitable for detection of BRAF35 by immunofluorescence and comes as 200ul, which will permit countless staining experiments.

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