I am trying to isolate the SlpA (Surface Layer Protein A) protein from Lactobacillus brevis with the Laemmli cell lysis buffer. but i am unable to get protein band in SDS PAGE run at the 44.31 KDa. Please suggest me is there any specific method to isolate the surface layer proteins. please suggest me what is the best way to purify that protein
Determine the protein concentration before running the sample. If the brotein is very low use sliver nitrate staining
Did you boil the sample?. Incubate the sample in lysis buffer for 45 minutes without boiling (only room temperature). The protein denaturation is slower and do not interact by forming precipitates.
Would be worth checking the strain you are trying to isolate. A good reference is http://www.ncbi.nlm.nih.gov/pubmed/17544232
http://www.ncbi.nlm.nih.gov/pubmed/17544232
Try brief (15 to 30 seconds) treatment with 15% ethanol at 25 and 37 C to release loosely bound proteins from membrane surface. After electrophoresis try treating the gel with 0.5 M ice-cold KCl. Use Try to vary it between 43-50 kDa. Its sometimes difficult to isolate Slps by the SDS-PAGE analyses, so you can also try PCR or partial sequencing.
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