Unusual source of contamination (brain protein extract SDS-PAGE)

Problem: I am not able to get good bands for the rat brain samples. It looks leaky and inconsistent as seen in the picture. I followed the exact protocol for kidney, liver samples and it worked. But it is not working for brain samples. I extracted the histone proteins using pre-lysis, lysis, and balanced buffer from Bio-Rad. Then checked the protein concentration followed by SDS-PAGE (80V for 1.5 hours). Then I did the transfer ( for 1.5 hours) on PVDF. I used H3K9BHB primary antibody (1:1000)dilution and kept in in cooler for 12-16 hours followed by secondary antibody (Anti-goat Rabbit 1:10000 dilution) for 2 hours. I developed the blots using LICOR’s fluorescence-based detection. Solution: To remove lipids from protein samples before performing SDS-PAGE, you can follow the steps below: Organic solvent extraction: One common method is to perform an organic solvent extraction using a mixture of chloroform and methanol. Mix your protein sample with an equal volume of the solvent mixture and vigorously shake the mixture. This allows lipids to partition into the organic phase. After centrifugation, carefully remove the upper aqueous phase, which contains your protein. If necessary, repeat the extraction step with fresh solvent mixture to further remove lipids. Detergent-based extraction: Another approach involves using detergents to solubilize and remove lipids. Add an appropriate detergent, such as Triton X-100 or NP-40, to your protein sample at a concentration below the critical micelle concentration (CMC). Incubate the sample with gentle shaking to allow the detergent to interact with the lipids. Following incubation, centrifuge the sample to pellet any insoluble material, and collect the supernatant containing the protein. Precipitation methods: Some lipids can be removed by protein precipitation techniques. Commonly used methods include acetone or ethanol precipitation. Add a volume of ice-cold acetone or ethanol (typically 4-5 times the volume of your protein sample) to the sample, mix thoroughly, and incubate at a low temperature (e.g., -20°C) for a sufficient period (e.g., 1 hour or overnight). Centrifuge the sample at a high speed to pellet the precipitated proteins, and carefully remove the supernatant. Dialysis or desalting: After removing lipids, it’s important to exchange the buffer of your protein sample to a suitable buffer for SDS-PAGE. This can be achieved by dialysis or desalting using appropriate molecular weight cutoff (MWCO) membranes or columns. Dialysis involves placing the protein sample in a dialysis membrane bag and allowing small molecules, including detergents and salts, to diffuse out while retaining the protein. Desalting columns use a resin or gel matrix to bind and exchange the buffer, effectively removing unwanted substances. The choice of method may depend on the specific characteristics of your sample and the nature of the lipids present. Additionally, it’s crucial to optimize and validate any lipid removal method to ensure that it does not adversely affect the integrity or activity of your protein of interest. #dna #gelelectrophoresis #genetics #pcr #biology