troubleshooting tips of IP, western and immunostaining

Normally when doing a westernblot we use a rainbow colour marker. We have been experimenting with an ECL colourmarker for we are now working with a chemi doc. Colourmarker tags cannot been seen on the picture the chemi doc takes and ECL markers should. We have used markers from Amersham Biosciences but after serveral tests we have not seen any markers yet. Does anyone have expiercience with these kind of markers?? Perhaps knows one that works in their lab? Or has a tip on why we haven't been able to get any makers.



With kinds regards



Judith de Vos
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I am trying to compare certain proteins of different tissues by using western blot. I can detect them very well but definitely I cannot get the same amount of protein in each lane. I've made extracts in the same conditions, I've dosed and I've normalized. Does somebody know how to do better?
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I am trying to compare certain proteins of different tissues by using western blot. I can detect them very well but definitely I cannot get the same amount of protein in each lane. I've made extracts in the same conditions, I've dosed and I've normalized. Does somebody know how to do better?
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I am trying to localize a virus in a mite. The problem I am having is non-specific binding of the secondary antibody (conjugated to FITC), leading to strong false-positive signals in my negative control and no signals in my positive. I fixed my mites with picric acid and I am worked with paraffin sections.
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I used western blot to discern positive and negative tissues for my protein of interest. I immunodetected this same protein in paraffin sections from these same cases and saw the exact opposite in staining pattern ie the positive tissues by western blot showed negative staining and the negative tissues by western blot were positively stained. How can this be?
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I used icc techniques to detect cytoskeleton of mouse oocyte and embryos using E7 and FITC-conjugated igG and DAPI to stain the nucleus. My problem with DAPI; the background was colored blue instead of the nucleus. With FITC, the backgound as well as the antigen were both green color but at certain points yellow color were also seen in some of the specimens. Your advise pertaining to this matter is highly appreciated.

Thank you

Best regards,

Nooraain
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Our lab uses immunohistochemical techniques to selectively stain particular membrane receptors on 4 micron thick mouse brain sections. We are much difficulty with uneven staining on these sections. Any suugestion on causes or prevention would be greatly appreciated. If further information is needed about our protocol please respond.

Thank you

Omar Saeed
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