Novex™ 18% Tris-Glycine Mini Protein Gels, 1.0 mm, 10-well

There may be too much beta-mercaptoethanol (BME), sample buffer salts, or dithiothreitol (DTT) in your samples. If the proteins are over-reduced, they can be negatively charged and actually repel each other across the lanes causing the bands to get narrower as they progress down the gel.

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Gel straightness is defined as the straightness across all lanes of the gel, measured at the bottom, expressed relative to the total length of the gel. For example, a gel with straightness of 0.020 Rf is flat to within 2% of the length of the gel (1.6 mm) across. Band curvature is defined as the curvature of the bands in the outer lanes of the gel, expressed relative to the total length of the gel. For example, bands with curvature of 0.010 Rf are straight to within 1% of the length of the gel (0.8 mm).

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The QC of our gels includes several processes:

1) Each gel is checked by eye for visible anomalies.

2) Under defined conditions, gels retained from each lot are tested as follows:

--When gels are run at a defined voltage, the resulting current and power of the electrophoresis are measured.

--Protein samples are electrophoresed on test gels to determine the gel run time and the protein band quality after electrophoresis. Bands are examined for: straightness within bands, curvature of bands across the gel ("smiling" or "frowning"), and reproducibility of the Rf values for protein molecular weight markers. According to these results, a Certificate of Analysis is created, which is available upon request.

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The most common cause of abnormally high current is the transfer buffer. If the transfer buffer is too concentrated, this leads to increased conductivity and current. High current may also occur if Tris-HCl is accidentally substituted for the Tris base required in the transfer buffer. This will again result in low buffer pH and lead to increased conductivity and current and subsequently, overheating. We recommend checking the transfer buffer and its reagent components and re-diluting or remaking the buffer.

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Here is the transfer protocol optimized for the Bio-Rad Semi-Dry Transfer Unit. NuPAGE transfer buffer can be used for transfer of Tris-Glycine gels.

- Working transfer buffer: 10% methanol, 1:1,000 Antioxidant in 2X NuPAGE transfer buffer (Bis-Tris 50 mM and Bicine 50 mM). If you need to prepare 100 mL of the working buffer from the NuPAGE 20X Transfer Buffer (Cat. No. NP0006), mix the following: 10 mL of 20X transfer buffer, 10 mL of MeOH, 100 µL of Antioxidant, 80 mL of DI H2O.

- Filter papers: The transfer buffer-soaked filter papers of the sandwich are the only reservoir in the Semi-Dry Transfer Cell. If Invitrogen pre-cut membrane/filter sandwiches are used, at least 2 extra filter papers (0.4 mm/filter in thickness) on each side of the gel (or membrane) are required. When assembling one gel/membrane sandwich, presoak 6 Invitrogen filter papers (or 2 thicker filter papers) and 1 membrane in working transfer buffer (prepared in step 1) and sandwich them on the top of the anode plate as follows: filter paper--filter paper--filter paper--membrane--gel--filter paper--filter paper--filter paper

- Blotting conditions: We found 15 V for 15-30 min is optimal for NuPAGE transfer buffer in the Bio-Rad Semi-Dry Transfer Cell. Semi-dry transfer units from other manufacturers should be used according to unit's instructions.

- For transfer of large proteins (100 kDa or larger), pre-equilibrate the gel with 0.02-0.04% SDS in 2X transfer buffer without methanol for 10 min before assembling the sandwich. Please note that transferring Tris-Glycine gels using NuPAGE transfer buffer in the Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell may be less efficient than using Tris-Glycine transfer buffer (Cat. No. LC3675) in the XCell II Blot Module (semi-wet).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.