HER2 Antibody for Immunofluorescence/Western blot

April 06, 2015

Oregon Health and Science University
Biomedical Engineering
Graduate Research Assistant

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Company:

Cell Signaling Technology

Product Name:

HER2/ErbB2 Antibody

Catalog Number:

2242

Cell Signaling HER2 antibody #2242 was used for both Western blot and immunofluorescence microscopy to determine the level of HER2 silencing by siRNA. This antibody was previously used to detect HER2 expression level in a panel of breast cancer cell lines with great success. For this experiment, HER2 positive breast cancer cell line BT474 was seeded either in a 6-well plate for Western blot or a 96-well plate for immunofluorescence. Transfection was carried out using Dharmafect 1 transfection reagent (Thermo Fisher) and 10nM of HER2 siRNA (Qiagen). Three days after transfection, protein was harvested using RIPA buffer from the 6-well plate and cells in the 96-well plate were fixed using 4%PFA. IRDye800CW conjugated secondary was used for detection on the Odyssey IR (Licor Biosciences) scanner for western blot. Alexa Fluor 647 conjugated secondary antibody was used for imaging on a EVOS Fl Auto fluorescence microscope (Life Technologies). Our results showed that 10nM of HER2 siRNA was effective in reducing HER2 expression by over 75%.

Experimental Design and Results Summary

Application

WB / IF

Starting Material

HER2 siRNA transfected BT474 cell lysate or fixed cells

Protocol Overview

WB: Cell lysates were run on a 4-12% PAGE gel followed by transfer to an Immobilon FL membrane. After blocking for 1 hour at room temperature with Licor Odyssey blocking buffer, overnight incubation at 4 degrees Celsius was done with 1:1000 dilution of HER2 antibody #2242 in blocking buffer. One hour incubation at room temperature using IRDye800CW conjugated goat anti-rabbit secondary antibody diluted 1:10,000 in blocking buffer was used for detection. Membrane was scanned on the Licor Odyssey IR scanner. IF: Cells were fixed in 4%PFA and then blocked with 2%BSA. A dilution of 1:200 was used for HER2 antibody #2242 in 2%BSA and incubation was done overnight at 4 degree Celsius. Alexa Fluor 647 goat anti-rabbit secondary antibody at a dilution of 1:200 was used for incubation at room temperature for 1 hour. Imaging was done on an EVOS FL Auto fluorescence microscope.

Tips

Works well for IF even though cell signaling did not label as such.

Results Summary

HER2 siRNA used in this experiment reduced HER2 protein level by over 75% in BT474 cell line. Antibody #2242 was specific for HER2 in IF and WB. No non-specific band was detected on WB membrane. IF signaling was mainly localized to the cell membrane.

Additional Notes

None

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Summary

The Good

High specificity for HER2 in IF and WB

The Bad

IF signal is not as bright as other HER2 antibodies

The Bottom Line

HER2 Antibody #2242 is very reliable and has minimal batch to batch variations. We have purchased at least 6 to 7 vials over the past 4 years and they all performed equally well.

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