22 replies to this topic

[eldon]

Hi Friends,

I am lysing the QT6 cells transfected after 48 hrs and doing my SDS-PAGE and western blotting. My protein has the HA antigen tag and i use 1:1000 dilution of HA monoclonal Ab.

To troubleshoot this problem v 2 people tried with the same blot, the same procedure and the same buffers. But i am getting again with high background. But the other person result was with clear band (only my protein of interst) without any bg.

It is difficult to identify where things r going wrong with my handling.
brief steps:
1) 1 hr blocking with 5% skim milk prepared in 1X TBS-T(tween 20 0.05%)
----after this, mild wash with 1X PBS-T to remove traces of skim milk.
2) 1 hr primary ab(1:1000) prepared in 1X TBS-T
3) wash 3 times with 10 min duration each with 1X TBS-T
4) 1 hr Sec ab (1:1000) prepared in 1X TBS-T
5) wash 3 times with 10 min duration each, with 1X TBS-T
6) ECL -Amersham Reagent and developing.

Expecting a reply
raje.

PVDF membranes? Make sure they are correctly activated with methanol...not doing so will give black background.

is primary polyclonal? try larger dilutions if yes...1:5000 - 1:10000.

1:1000 secondary is HRP conjugate? This sounds too high. 1:5000 to 1:20000 depending on quality of the primary.

Wash more at all washing steps. Heed the advice already offered about "pre-washing" extra Ab before your timed washes. I have never over-washed a blot in my life and I'll wash sometimes 4-5 changes 20 min. each only because I am running too many other experiments at the same time.

[DaRQsiDe]

Hi,

If your lab has money, you should get a good western blotting kit - save yourself the headache. I have been using the GenScript One Hour western kit for years and it never failed me.

Otherwise, don't use milk, use 1.25mg/ml BSA in TBST or PBST with around 0.5 to 1M NaCl.

[liweixie]

Increase washing time would be helpful to reduce the background

[Boba]

many suggestions have already been made. Try to increase the concentration of your tween-20 by half a percent. you should def. incubate your primary antibody in milk...do more frequent, higher volume washes and shake aggressively.

[bkbala]

Hai

[bkbala]

Hai all
This is first time I am doing westernblot. I have doubt about the loading control and I don't know how to use the loding for WB. Could anybody help me in this aspect.

[PDGF]

many suggestions have already been made. Try to increase the concentration of your tween-20 by half a percent. you should def. incubate your primary antibody in milk...do more frequent, higher volume washes and shake aggressively.

In general, I would recommend using a kit. I use the 30-minute kit from Medhus Bio; its a little less sensitive than a normal western blot but almost makes the process a high throughput one. The popular ECL kit is a little expensive, but still quite good.

Using fresh tween-20 can help as well.

I would recommend PBST w/BSA over milk for blocking.

[js86]

Hi Friends,

I am lysing the QT6 cells transfected after 48 hrs and doing my SDS-PAGE and western blotting. My protein has the HA antigen tag and i use 1:1000 dilution of HA monoclonal Ab.

To troubleshoot this problem v 2 people tried with the same blot, the same procedure and the same buffers. But i am getting again with high background. But the other person result was with clear band (only my protein of interst) without any bg.

It is difficult to identify where things r going wrong with my handling.
brief steps:
1) 1 hr blocking with 5% skim milk prepared in 1X TBS-T(tween 20 0.05%)
----after this, mild wash with 1X PBS-T to remove traces of skim milk.
2) 1 hr primary ab(1:1000) prepared in 1X TBS-T
3) wash 3 times with 10 min duration each with 1X TBS-T
4) 1 hr Sec ab (1:1000) prepared in 1X TBS-T
5) wash 3 times with 10 min duration each, with 1X TBS-T
6) ECL -Amersham Reagent and developing.

Expecting a reply
raje.

hello friend
firstly increase ur washing time.
and if 1:1000 dilution is not recommended for 2ndary antibody use its 1:10000 dilution.
washing step is important in case of backgrounds
hope so it will work..