Works Well for Western Blot

Department of Biosciences
Rice University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

MiliporeSigma

Product Name:

Anti-Sulf2, Clone 2B4

Catalog Number:

MABC584

This product was used to investigate the expression of SULF2 across a number of different cell lines via western blot. Here we show how the antibody performed on lysate from MCF-7 cells.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Human MCF-7 Cell line lysate

Primary Incubation

Anti-SULF2 was used at 1 µg/mL in blocking buffer, overnight incubation at 4°C.

Blocking Agent

PBS 0.1% Tween20, 5% Blotting grade Non-fat dry milk, 1 hr incubation at RT

Secondary Incubation

Secondary AB Sheep anti-Mouse was used at 1:50000 diluted in blocking buffer, incubated for 1.5 hrs at RT

Tertiary Incubation

N/A

Detection

ECL substrate SuperSignal West Femto Max Sensitivity

Results Summary

MCF-7 cells were treated with either scrambled or SULF2 siRNAs for knockdown of SULF2. After knockdown, lysate was collected from both groups using RIPA buffer with protease inhibitors (Halt) and protein concentration measured using BCA assay. 30 µg of lysate was loaded per lane of a pre-cast 10% bis-tris gel, which was run at 200 V using a MOPS SDS running buffer for 50 minutes. Transfer was done using 20X NuPage Transfer Buffer at 40 V for 5 hours at 4°C. Exposure time was 2 minute. Three bands were picked up by the antibody, ~80 kDa, ~95 kDa and ~125 kDa. All of which were not present in the knockdown (KD), and only the ~125 kDa (weakest) was predicted by the company. We are not sure what the other bands are.

DOI or PMID #

N/A

Additional Notes

N/A

Related Categories

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Summary

The Good

Seems to work, but will require optimization for your cell line

The Bad

Non-specific bands may be detected

The Bottom Line

It's an OK antibody but bear in mind you will need to optimize your system and cell line of choice.

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