If anyone can help, I am interested in performing antigen retrieval on a membrane bound protein from formalin fixed tissue to use to detect in a Western Blot. The tissue has not been fixed in paraffin and is frozen in a 3.7% formaldehyde (10% formalin). I am using a RIPA buffer (0.1% SDS) so I have also had trouble with my protein quantification assays (Bradford and BCA). I have been fixing brain tissue in formalin for 24 hours at RT and freezing the tissue until needed. After, thawing in 4 degree C fridge I tried rinsing with tap water for a half hour or so and then 2 x washes with 100% ethanol. I submitted samples in either sodium citrate (pH=6.0) or TBS-T(pH=9.5) to 100 C water bath for 20 mins and 25 mins. After warming to RT, I removed samples and transferred them to 300 micro liters RIPA and 180 micro liters protease/phosphatase inhibitors. I mechanically homogenized the tissue with 1.0 mm boron silicate beads for 5 mins. Next, I centrifuged for 20 mins. at 12000 G. I aspirated the supernatant and transferred to other eppendorf tubes and froze in -40 C freezer until western. SOrry the long intro. After this I ran a 1D Western blot and had streaking lanes. There may have been some banding but the streaking was so bad I can't really visualize. I ran this before and had solid banding at correct kD. After talking with a friend I think not rinsing properly after removal from high and low pH buffers, that it may have affected the gel chemistry. So hopefully this wasn't too confusing, but here are some questions I now have:
1. Is there a range of pH that the gel in gel electrophoresis operates in and can even minute changes in pH make the gel streak?
2. I am trying antigen retrieval and it primarily is used in Immunohistochemistry and not WB. The protocol I have found is for IHC, so in my buffers I heated the samples in, I added Tween 20 to my buffers as the protocol called for, but am unsure if this should be done for WB since the IHC antigen retrieval calls for placing tissue in blocking solution containing Tween 20 following heating in buffer.
3. If you are wondering why I am doing this, it is because we have frozen brain tissue fixed in formalin.