Myocilin, R&D Systems, MAB3446

This antibody was included in a study aiming to characterize and validate commercially available antibodies raised against myocilin. Epitope mapping was conducted using recombinant myocilin subdomains cloned and expressed in E. coli. As positive control was used endogenously expressed full-length human myocilin secreted by trabecular meshwork (TM) cells.

We evaluated four antibodies (Ab41552, MABN866, MAB3446, sc-137233) for application in Western blotting (denaturing conditions) and ELISA (native protein).

Our data show that MAB3446 recognizes an epitope within the amino acid sequence 112-185 (the LZ subdomain) of myocilin. The antibody works very well for WB, and detects both E. coli expressed myocilin fragments containing these residues as well as the full-length human protein.

The epitope was confirmed by ELISA.

For further information see Patterson-Orazem et al., 2018 “Epitope mapping of commercial antibodies that detect myocilin” (www.ncbi.nlm.nih.gov/pubmed/29752947)

Anti-Heterochromatin Protein-1 α (HP1α), Mouse Monoclonal Antibody, Clone 2HP-2G9, EMD Millipore, MAB3446

The antibody was used for Western blotting in order to detect knockouts of Heterochromatin Protein 1α (HP1α, approx. 22 kDa).

As seen on the picture (Western blot after 4 min exposure) it binds well to the control sample and also disappears in the wells loaded with protein from knockout colonies, but there is a lot of non-specific binding (Bands all over the place).

It was used for in a dilution of 1:1000 in 10 mL washing buffer in a 50 mL tube.

20 µg of protein solution purified from MCF7 was used per well for the Western blot

Protocol:

20 µg of purified protein extraction procured from 1 control and 6 mutant MCF7 colonies were prepared in appropriate amounts of loading buffer under the fume hood and heated at 100 oC for 10 min.

The marker used was quickly heated to 37 oC and samples + marker were loaded and run on a Mini-PROTEAN® TGX TM Precast gel from Bio-Rad for approx. 30 min at 40 mA.

The bands were blotted for 30 min (60 V, 0.5 A) to an Amersham PVDF membrane from GE Life sciences under cooling conditions.

The membrane was blocked O.N. in 4% blocking buffer (2 g Difco skim milk powder, 5 mL 10% tween 20 diluted to 50 mL in PBS) in a refrigerated room.

The membrane was then incubated with the primary antibody:
Monoclonal anti-HP1α (mouse) purchased from EMD Millipore (MAB3446) diluted to 1:1000 in 10 mL washing buffer (5 g skimmed milk powder, 10% tween 20 diluted to 1 L with PBS) in a 50 mL tube. The tube was placed on a rotor in a refrigerated room for 1.5 hour.

The membrane was then moved to a plastic box washed 3x 5 min in washing buffer on a tilting table, before removing the washing buffer and adding the secondary antibody: polyclonal anti-Mouse (goat) from Dako diluted 1:10,000 in 20 mL washing buffer.

The box was covered in foil and incubated an additional hour on the tilting
table.

The membrane was washed 5x 5 min in washing buffer on the tilting table and then covered in development solution (10 mL Blotting substrate and 100 µL Starting solution) for 1 min.

The membrane was quickly dried, put in a plastic pocket and transferred to a Kodak cassette.

The membrane was developed on x-ray film in a AGFA curix60 film processor in the dark room.

The assay was repeated with freshly ordered antibody and also 10% blocking buffer instead of 4% in order to try and reduce non-specific binding, but the same pattern arose as previously (See picture).