SDS-PAGE Electrophoresis

1.Uneven glue? Gel leakage?

· Clean the rubber plate.

· The amount of APS and TEMED should be appropriate.

· After adding reagent, shake well to make it fully mixed to prevent uneven polymerization of some rubber blocks.

· Proper temperature and uneven heating lead to uneven polymerization.

· The bottom of the two panels should be aligned.

2.Is the band narrower than normal?"Smile" or "back smile" band?

· Gel polymerization is uneven, when glue is used, mix evenly as possible and act gently.

· The comb should be pulled out quickly, and the sampling hole should be cleaned carefully to avoid twisting the sample belt.

· If the salt concentration of the sample is too high, other bands will be squeezed, resulting in different width. Purify the sample and adjust the salt concentration.

· Bubbles at the bottom of the rubber plate will affect the electrophoretic effect and should be driven away. At the same time, pay attention to whether the electrophoresis tank device is suitable.

Membrane transfer and antibody detection

1.Swelling or curl of gel?Strip skew or drift?Single or multiple white dots?Membrane transfer buffer overheated?

· The gel can be soaked in the film buffer before being transferred to 5-10min.

· The sponge became thinner after long-term use of the electric rotameter, and the "sandwich" structure was not compact.You can put a little common straw paper between the two sponges.

· Make sure there are no bubbles between the film and the rubber block.

· The ion concentration in the buffer is too low and the current or voltage is too high. Pay attention to temperature reduction during membrane transfer.

The background is too high

Reason

1.The membrane was not evenly wetted.

2.Membrane or buffer contamination.

3.Inadequate closure.

4.There was cross reaction between antibody and sealant.

5.Antibody concentration too high.

Countermeasure

1.The membrane was completely soaked with 100% methanol before conversion.

2.When taking the film and absorbent paper, wear gloves and replace the fresh film transfer buffer.

3.The cross reaction between the first antibody, the second antibody and the blocking agent was detected.

4.The working concentrations of the first and second antibodies were detected before hybridization.