Blotting Techniques

Query regarding small size protein transfer on Nitrocellulose membrane ( pore size- 0.45u).

My protein is of molecular weight below 20 kDa. I transferred it for 30 min at 90V but could not see distinct bands i.e. smear type on Ponceau staining.15% gel was used and was run half of the...

12 December 2013 9,432 3 View

Can I transfer a 6KDa protein with nitrocellulose membrane (0,2 um)? Does anyone know how to deal with such low molecular weight proteins?

I'm experimenting with some protocols to blot EGF protein. Do you have any tips to handle EGF in Western Blot assay?

12 December 2013 4,127 5 View

Can someone suggest a simple method of Genomic DNA extraction from Actinomycetes?

I am planning to extract Genomic DNA from Actinomycetes, and I don't need a pure DNA. I am NOT planning to pcr the DNA. What is a simple method, that does not involve DNA extraction kits or something?

11 November 2013 1,737 4 View

Why do we need to blot the DNA-protein complex in an enzyme mobility shift assay?

I am doing EMSA and I have question. Why do we need to transfer the DNA-Protein complex on nylon membrane? Why can't we dry the gel? If anyone has the answer for this please let me know.

06 June 2013 4,759 2 View

How to isolate proteins from PVDF blotting paper for MALDI-TOF analysis?

Does anyone have experience with identification of protein from blotting membrane? I tried to follow several methods, but I have found very less robustness and high protein loss during the...

11 November 2012 5,130 9 View

Why add tween 20 0.1 wt% in tris-buffered saline during the western blotting?

I am not make sense the reasons for adding tween 20.

01 January 1970 3,474 3 View

Some one can help me with some topics on southern blotting?

Hi everyone. I'm doing right now some southern blotting assays on a plant genomic DNA and several questions have arised. 1. Why is it necesary to digest over night? I had seen a simmilar pattern...

01 January 1970 9,883 1 View

Which exosomal marker for Western Blot

Hi everyone, I am working on secreted peroxiredoxins (PRDXs) from RAW 264.7 cells and have various Western blots of cell supernatants which I have already blotted for PRDXs. These proteins are...

01 January 1970 9,641 2 View

I am unable to obtain an occludin signal via WB on my rat brain samples. Is there any specific protocol to detect occludin?

Rat hippocampus homogenized in RIPA buffer with P/P inhibitors.  BCA. Heated 70 degrees for 10 min.  Run on 4-12% bis-tris gel with MOPS buffer.  7 Minute xfer with trans-blot turbo.  Occludin...

01 January 1970 6,368 3 View

Imunopercipitation western bloting?

what is the different between immunoprecipitation-western blot and western blot? For measurements of proteins, that contributed in necroptosis, such as RIP-1/3, immunoprecipitation- western blot...

01 January 1970 4,115 4 View

No title

01 January 1970 6,680 0 View

How to remove protein from western blots?

Dear all, I have been having problems with various antibodies in my western blots. For some reason they remain on the blot even after they were washed off (0.5% TBST 3x 5 minutes). I have tried...

01 January 1970 595 3 View

Why add tween 20 0.1 wt% in tris-buffered saline during the western blotting?

I am not make sense the reasons for adding tween 20.

01 January 1970 2,429 2 View

No title

01 January 1970 1,190 0 View

I am unable to obtain an occludin signal via WB on my rat brain samples. Is there any specific protocol to detect occludin?

Rat hippocampus homogenized in RIPA buffer with P/P inhibitors.  BCA. Heated 70 degrees for 10 min.  Run on 4-12% bis-tris gel with MOPS buffer.  7 Minute xfer with trans-blot turbo.  Occludin...

01 January 1970 9,955 3 View

No title

01 January 1970 7,291 0 View