2 replies to this topic

[9939]

Hi all,

I'm having some problems with WB using post-transfected cell linea. The bands showed on my WB doesnt correlate with the expected band size. =(

1. I designed a gene construct (let's call it plasmid P here) tagged with HA, send for sequencing (the result was fine).

2. I cultured cells in 10cm plate and transfect them with plasmid P using lipofectamine 2000 from Invitrogen according to manufacturer's instructions.

3. Upon 48 hours, I collected the cells with RIPA buffer (50mM Tris-HCl pH8.0, 150mM NaCl, 1% NP-40. 0.5% Na deoxycholate, 0.1% SDS) . Methods of collection is as followed:
- Aspirate media. Wash twice with 1X cold PBS.
- Add 1ml of ice cold RIPA buffer, incubate on ice for 5 mins.
- Collect the cells via scraping, collect it on tube.
- Centrifugation at 8000g for 10min at 4deg.
- Collect supernatant. Did protein concentration measurement. Store at -80deg.

4. WB
- Sample preparation- Add 5X loading buffer (1.25ml Tris 0.5M pH 6.8, 2ml glycerol, 2ml 10% SDS, 0.5ml B-mercapthanol, estimation of bromophenol blue, top up to 10ml total)
to samples. Boil for 5mins at 95deg. Total protein loaded is 20ug protein per well.
- Run normal WB.
(Note: The primary Ab used is of goat polyclonal origin.)

The attached files shows WB slides for my protein of interest (~43kD) and the control beta-actin. Loading sequnce:
1. Plasmid P transfected cells
2. Plasmid P transfected cells (as I was trying different conditions of transfection)
3. Non-transfected cells

I've uploaded the scanned film (protein of interest and actin). Nonetheless, it was shown on the protein of interest film that that there are multiple bands, and none of them is of 43kDa!

Sorry that the film looks dirty (I've changed the secondary Ab used after series of experiment with dot blot and I'm running a new set of WB now).
But the probblem is, the bands size shows is not what I was looking for. =s

Some of the reasons that I can think of (but I'm not sure of):
1. Non-specific binding of Ab (both primary and secondary).
2. Preparation of protein samples was inadequate (denature? disintegrate?)
3. Problem with transfection (not very sure what could go wrong there).
4. Tagged protein will have different size?? (??)

Does anyone have any comments on what could have go wrong here? Any suggestions are highly appreciated. Thanks!

[madrius1]

That seems pretty good to me..

First of all, you can see a major band appear in both transfection conditions, whereas no such band is present in the non-transfected lane. In cell line A and B. Moreover, the major band is pretty close to 50, but seems to be smaller than 50. If you expect a band at 43, and you have a fusion protein tagged with HA running close to but still under 50, the band you see is most certainly the good one. So in my opinion, you have a working plasmid my friend!

I can still answer your questions, though.

1. For the HA blot, the minor bands/background are a direct proof of non-specific binding of you Abs.
2. The actin blot is really staight, what makes you think of inadequate preparation or degradation? Don't you think it would have had consquences on the actin as well as your protein?
3. Given the points i've mentionned, the transfection seems to work very well.
4. Absolutely.

Edited by madrius1, 20 January 2010 - 11:05 AM.

[bob1]

Also - probe for your protein of interest on a new blot - you should see brighter bands in the transfected lanes, as well as a doublet if you get the exposure right - the bigger band will be the HA tagged protein, the smaller is the native.