I need to compare level of expression of a protein in E.coli cell lysates. In order to do so we want of take equal amount of protein from cell lysates. But I have too many samples to be processed by biochemical assays like Bradford etc.
We ended up normalizing the UV280 absorbance of all samples by fixing the absorbance at UV280 to 0.5.
My query is regarding the usage of using same UV280 as a measure of protein amounts to be loaded for western. Has anyone been following the practice?
Someone on Research Gate answered this question 5 years ago.
Uttam Pal stated that only 3 amino acids Tyr, Tryp and Phe
can absorb UV light. Here is what he said:
" Only three amino acids (Trp, Tyr, and Phe) have conjugate system so they absorb UV light. But Phe absorption is negligible. Therefore you can only measure Trp and Tyr. The absorption maximum for Tyr and Trp is about 280 nm. They also fluoresces. You can measure their concentration from the absorption data at a particular wavelength using their molar extinction coefficient at that wavelength (use this formula OD = e.c.l; where e is the molar extinction coefficient, c is the concentration, l is the pathlenght of the cuvette used)."
My advice? Get access to a plate reader and do Bradfords.
If all the samples have essentially the same composition and were prepared in the same manner, only differing in the concentration, then using an A280 measurement to figure out how much to load on a gel seems reasonable. However, if the samples are of different compositions or were prepared in different ways, then this approach may not be very accurate. A protein assay might be better.
You could Ponceau S stain the membrane prior to probing it to check for similar loading. You could also co-probe the blot with an antibody to a housekeeping protein as a loading control.
Hello,
Regardless the samples you are working, a better way to compare the protein expression, I suggest you go through OD600 measurement. Since the proteins are in cells, the UV280 or bradford may not be effective. Use following steps:
1. Take OD600 measurement of all samples
2. Normalize all the samples to OD 4 by dillution or concentration using centrifuge. You can use MiliQ water to normalize or if you have media (YT, TB, LB), you can use them
3. Use lysis buffer to lyse the cells.
4. Seperate the soluble or insoluble
5. Load samples to GEL. For OD 4, you can load 5-15 microliter depending on the level of expression.
6. If you have lots of samples, just use to normalized sample and perform SDS-PAGE.
I hope this will work.
Hi Adam,
Yes, the samples are identical and have been prepared by same lysis method in the same batch. To add up to the consistency..i do not compare between batches.
I will use Ponceau t stain the blot.
Thanks
Hi Pradeep,
The measurements at OD600 would help me take equal amount of cells as a starting material. This would be similar to taking a wet weight of cells before the experiment to normalize the amount of cells.
But in order to accomodate for differences in the lysis efficiency or any loss during the processing wouldn't it be useful to measure the A280?
- Badges
- Science method