Sage-Tips

What causes smearing on SDS-PAGE?

Smearing can have a variety of causes, but most commonly it is due to an unevenly poured acrylamide mixture or due to gross overloading of protein. In this example the gel was not properly poured, so that the lower half had begun to polymerize before the upper part was poured.

What is the most common material to prepare SDS-PAGE gels?

For the gel solution, acrylamide is mixed as gel-former (usually 4\% V/V in the stacking gel and 10-12 \% in the separating gel), methylenebisacrylamide as a cross-linker, stacking or separating gel buffer, water and SDS.

When should I stop running SDS-PAGE gel?

When the dye front is nearly at the bottom of the gel it is time to stop the run. For low percent gels with a tight dye front, the dye should be on the verge of running off the gel.

How do you prevent smearing in Western blot?

Western Blot possible causes & solution for smeared bands Titrate down the amount of protein loaded per lane. To ensure sharp banding, always use fresh APS and TEMED, and allow at least 30 minutes for the gel to polymerize before running. The optimal voltage is mostly determined by the apparatus used.

Why is my Western blot smeared?

Smearing on Western blots can be caused by non-specific binding of the antibody, insufficiently tight contact between the gel and filter during transfer, mishandling of the filter after transfer, and low signal-to-noise caused by weak detection.

Is SDS-PAGE a Western blot?

SDS-PAGE is an electrophoresis method that separates proteins by mass. Western blot is an analytical technique to identify the presence of a specific protein within a complex mixture of proteins, where gel electrophoresis is usually used as the first step in procedure to separate the protein of interest.

What is SDS-PAGE made of?

In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length.

How do you visualize SDS-PAGE gel?

Visualization of protein bands is carried out by incubating the gel with a staining solution. The two most commonly used methods are Coomassie and silver staining. Silver staining is a more sensitive staining method than Coomassie staining, and is able to detect 2–5 ng protein per band on a gel.

How much SDS do I need to make a gel?

10g SDS up to 100mL water Buffers for Making Gels Lower Buffer (for the separating gel) 1.5M Tris 36.4g pH to 8.8 with 6M HCl until nearing desired pH 0.4\% SDS 8mL of 10\% SDS up to 200mL with water Stacking (Upper) buffer (for the stacking gel) requires fair bit of HCl so start with ~70mL water when add Tris

How to make SDS polyacrylamide gels?

Making SDS Polyacrylamide Gels* (once the last ingredient, ammonium persulfate, is added, the gel will begin to polymerise) Separating (lower) Gel – 10\% acrylamide (change the proportions of water and acrylamide if different from 10\% acrylamide)

What to do if the gel is leaking from the bottom?

Last, as a desperate last attempt, when the gel is leaking from the bottom immediately add more of TEMED and APS directly onto the leaking mess! it will itself form a plug sooner than the gel will take to solidify, thus restricting further leakage. Before pouring in the acrylamide mix, pour in water to check for leaks.

What is the western blot protocol?

Western Blot protocol Making the gel 1. Use alcohol and Kimwipes to wipe the glass, and then set up the rest of the apparatus 2. For small proteins, use a higher percentage gel 2.