Distortion of bands
The most common complaint for Phos-tag™ SDS-PAGE is gDistortion of bands. Especially, make sure not to contain EDTA in your samples.
① Prestained marker: Not only the lane of the marker but also the sample lane may be affected due to a difference in the salt concentration among lanes.
⇒ Prestained markers should not be used. (See the right figure.)We recommend to use alkaline phosphatase-treated sample or recombinant protein of your target one as the negative control of phosphorylation instead of using prestained markers.
② Acidic sample: Bands may be distorted. If the solution is a yellow to orange color even after loading sample buffer, add Tris buffer until it is neutral (violet).
③ EDTA (Mn2+ is chelated), vanadic acid, inorganic salts, surfactants, etc., cause distortion or tailing of bands.
⇒ Desalinate by precipitation with TCA or dialysis.
④ Blank lanes: Blank lanes may cause distortion.
⇒ Load the same amount of 1 x sample buffer in blank lanes.
⑤ Vanadic acid: Competitive binding with phosphoric acid may cause distortion.
⇒ Use a different phosphatase inhibitor or remove vanadic acid by precipitation with TCA or dialysis.
⑥ Adding MnCl2 to the sample to be applied (eg: 1 mM final concentration) may improve results. If the sample contains an EDTA residue, it is because the added Mn2+ is chelated instead of the Mn2+ contained in the gel.
① Rise of the molar ratio of MnCl2 to Phos-tag™ Acrylamide may improve the resolution. (eg: 1F4)
② Adopting Tris-Tricine Buffer as Running Buffer may improve the resolution.
Long-term migration with a constant current will cause decomposition and diffusion of proteins due to excessive heat.
① If you want to use a constant current for migration, try techniques such as using a low-temperature room, thoroughly cooling the migration buffer just before use, and wrapping a cooling agent around the migration tank (but do not use ice because it may cause electric shock).
② When a constant voltage can also be used, migrate with a constant voltage (eg: 200 V). The migration speed will slow down but the generation of heat will be suppressed.
Easy breaking of the gel
The gel is softened due to the low concentration of acrylamide.
① 5% or higherFIncreasing the N,Nf-methylene-bisacrylamide to acrylamide ratio (eg: 24F1) will strengthen the gel.
② Add 3`5% of agarose to strengthen gels. Refer to gPreparing a Low-Concentration Gel Containing Agaroseh in g3. Protocolh and gSeparationh in g7. FAQ.h
Trouble with Transfer onto the membrane
① Treatment with EDTA may be insufficient. Increase the treatment time, frequently exchange the buffer containing EDTA (eg: 20 min. x twice), or fully agitate during EDTA treatment.
② To intensify the electric current may improve efficiency (eg: 200 mA)
③ Stainings other than negative staining such as CBB staining may diminish the transfer efficiency.
④ Using a low-concentration gel will improve the transfer efficiency.
⑤ Take steps such as using a thick gel and increase the sample application amount.
⑥ Transfer buffer containing SDS may improve the transfer efficiency. When transferring onto a membrane, immediately add the SDS solution to the transfer buffer to prevent sudsing (tank method), or between the EDTA process and transfer, immerse the gel in a transfer buffer containing SDS and shake slowly (eℊ: for 10 min. x 1 time). Try SDS concentrations of 0.05`0.20%.
Membranes after Transfer Sample: WIDE-VIEW™ Ⅲ Prestain Marker (Code No. 230-02461))
① No EDTA processing
② 1mM EDTA for 10 minutes x 2 times
③ 10mM EDTA for 10 minutes x 1 time
④ 1mM EDTA for 10 minutes x 2 times
12.5% Phos-tag™ Super Sepi50mMj
Marked transfer efficiency drop occurs without EDTA processing.
For a reliable transfer, 1 - 10mM EDTA process for 10 minutes x 2 times is recommended.
Higher background during staining
Stain after eliminating metal ions in the gel by EDTA treatment.
Mobility shift due to protein degradation, not induced by phosphorylation
Carry out SDS-PAGE as usual (containing 0 μM of Phos-tag™) and verify that no mobility shift occurs.