Our lab performs western blotting on a daily basis and we routinely stain the membrane with ponceau S red solution following transfer to determine transfer efficiency. Unfortunately, ponceau S red solution suffers from a lack of sensitivity and since ponceau S red specific staining is removed rapidly in aqueous solution it is difficult to accurately document the efficiency of protein transfer.
In a search for alternative staining methods we came across the MemCode staining kit for PVDF membranes from Pierce Biotechnology. This kit consists of four solutions; a sensitizer solution, a reversible staining solution, a de-stain solution and an eraser solution. The instructions state that there is enough solution to stain/de-stain 10 PVDF membranes if you use the proposed 25 ml per membrane. However, the protocol provided is easily modified so that a smaller volume of solution can be applied. We have found that dispensing 4 ml of each of the solutions directly onto a piece of saran wrap and placing the membrane on top, protein face down, works equally well. In addition, the sensitizer and staining solutions can be used for multiple blots with no notable loss of protein staining efficiency.
The reversible staining solution stains the membrane-bound protein turquoise and the subsequent de-stain step results in very low background. Importantly the de-stain solution removed only the background membrane staining without visible loss of protein band staining. In general, we found that an additional de-stain:methanol step mentioned in the protocol after the initial de-stain was not necessary since the de-stain steps alone allowed the visualization and documentation of very discrete protein bands. The eraser solution proved to be very effective at removing the stain completely from the membrane and generally required around a 5 min incubation period with constant and vigorous agitation. Furthermore, we were able to reuse the eraser solution for up to 4 membranes without any noticeable loss of the effectiveness of de-stain.
In summary, the MemCode PVDF membrane staining kit proved to be reliable, simple to use and resulted in superior staining of protein bands over the commonly used ponceau S red solution.
Jonathan M. Shillingford, Ph.D.
Research Associate
Dept of Cell Biology
Lerner Research Institute
Cleveland